Biochemical Characterization of Rat Intestine Development Using High-Resolution Magic-Angle-Spinning 1H NMR Spectroscopy and Multivariate Data Analysis Yulan Wang,*,† Huiru Tang,† Elaine Holmes,† John C Lindon,† Marco E. Turini,‡ Norbert Sprenger,‡ Gabriela Bergonzelli,‡ Laurent B. Fay,‡ Sunil Kochhar,‡ and Jeremy K. Nicholson† Biological Chemistry, Biomedical Sciences Division, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building, South Kensington, London, SW7, 2AZ, United Kingdom and Nestle´ Research Center, P. O. Box 44, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland Received February 14, 2005
We report details of metabolic profiles for small intestinal samples obtained using high-resolution magicangle-spinning (HRMAS) 1H NMR spectroscopy. Intact samples of jejunum and ileum from male Long Evens rats were analyzed on a 600 MHz spectrometer using standard one and two-dimensional 1H NMR spectroscopic pulse sequences. The metabolic profiles of ileum and jejunum predominately comprised a number of amino acids, lipids, glycerophosphocholine (GPC), choline, creatine, and ethanol, a number of carboxylic acids including acetate and lactate, and nucleoside bases including cytosine, isocytosine, and uracil. Principal component analysis (PCA) was applied to these NMR data to characterize the biochemical differences between jejunum and ileum tissues. Compared with ileum, jejunum contained higher levels of lipids, GPC, choline, lactate and creatinine, but lower levels of amino acids and acetate. In addition, the age dependence of the biochemical composition of intestinal tissues from young rats (15, 36 days and 3-4 months old) was studied. In general, levels of lipids, lactate, taurine and creatinine were positively correlated with age while amino acids and GPC decreased in the older age group. This study will provide a metabolic reference for further studies assessing the metabolic consequences of nutrition, stress and gut microbiota on intestinal composition. Keywords: metabonomics • development • jejunum • ileum • intestine • principal components analysis • HRMAS 1H NMR spectroscopy
Introduction An increasing awareness of the potential of gut microbes to influence human health has led to widespread investigation of the relationship between the gut microbiota and nutrients, particularly prebiotics and probiotics, and their impact on the digestive system.1 Thus, a renewed interest in the relationship between the structure and function of intestinal tissues has evolved. The small intestine is an important part of the digestive tract of mammals and has a crucial function in food digestion and absorption of nutrients into the blood stream. Generally, the small intestine can be divided into three parts, duodenum, jejunum, and ileum. The duodenum is connected to the stomach at one end and is where major digestion takes place aided by digestive enzymes excreted from the pancreas and other intestinal glands. The jejunum is located between the distal end of the duodenum and the proximal part of the ileum, although the transition from the jejunum to the ileum is not * To whom correspondence should be addressed. Tel: 44(0) 20 7594 3023. Fax: 44(0) 20 7594 3226. E-mail:
[email protected]. † Biological Chemistry, Biomedical Sciences Division, Faculty of Medicine, Imperial College London, Sir Alexander Fleming Building. ‡ Nestle´ Research Center.
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marked by any specific anatomical structure. Structurally, the two tissues can be easily distinguished as the diameter of the jejunum, especially the proximal part, is larger than that of the distal ileum. The wall of the jejunum is thicker and has more circular folds, hence providing a greater absorption surface. However, little information is available on the biochemical composition of different parts of the small intestine, or on their responses to influential factors such as age, gut microbiota, diet, or stress. The structure and functional integrity of the small intestine are largely influenced by diet,2 age,3 stress,4,5 and state of health.6 In addition, a large number of bacteria (typically 5001000 different species) are resident in the intestines as symbionts.7 Common bacterial species found in the small intestines include Lactobacillus, Bacteroides, Clostridium, Mycobacterium, Enterococci and bacteria of Enterobacteriaceae.8 In the human, the population density of such bacteria increases by ∼8 orders of magnitude from the proximal small intestine (103 organisms per milliliter luminal contents) to the colon (1011 per g of content).7 It has been well documented that indigenous microbiota exert a profound impact on the development and structure of the intestinal epithelium, the digestive and absorp10.1021/pr050032r CCC: $30.25
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NMR Spectroscopy of Rat Intestine Development
tive capabilities of the intestine and on the host immune system. For example, comparisons of rodents raised in germfree conditions with those raised in conventional conditions revealed that the microbiota directs assembly of the gutassociated lymphoid tissue,9 facilitates the education of the immune system,10,11 influences the integrity of the intestinal mucosal barrier,12-14 alters proliferation and differentiation of its epithelial lineages,7 changes the activity of enteric nervous system and has a profound impact on extracting and processing nutrients consumed in diet.15 Previous work on the biochemical composition of the small intestine has been published based on 1H NMR spectroscopy of perchloric acid extracts of rat small intestine samples taken during periods of ischaemia and reperfusion.16 However, the molecular information was limited and was highly dependent on the extraction method. In addition, extraction procedures are destructive and do not retain information on molecular compartmentation.17 These limitations can be largely overcome by high-resolution magic-angle-spinning (HRMAS) NMR spectroscopy of intact intestinal tissue samples. HRMAS NMR spectroscopy involves spinning tissue samples at the magic angle (54.7° between the rotation axis and the NMR magnetic field direction), and this causes the major line broadening factors such as residual dipole-dipole interactions, NMR chemical shift anisotropy and anisotropic magnetic field inhomogeneities to be averaged out.18 To achieve optimal spectra, the rotation rate must be greater than the observed line broadening and is typically ca. 4 kHz. The main advantage of this approach is that it can provide detailed molecular information on small intact tissue samples (∼15 mg), and generates a molecular signature that potentially encodes latent biological or pathological information.19-22 In addition, it is possible to study molecular dynamics, as compartmental information in the intact tissue is retained. HRMAS NMR spectroscopy has been applied to the characterization of intact rat liver,19,23,24 heart,25 kidney,26 testis,21 human breast,17 and prostate27 in healthy and diseased animals and humans. It has also been applied to the evaluation of the toxic effects of arsenic,22 cadmium,28,29 2-bromoethanamine,20 alpha-naphthylisothiocyanate30 in experimental animals. Recently, HRMAS NMR has also been employed to study the biochemistry of healthy human gastric mucosa and this revealed about 40 molecular species in that tissue.31 Here, we report the use of 1H HRMAS NMR spectroscopy in combination with principal component analysis (PCA)32,33 to characterize the biochemical composition of intact intestine tissues including jejunum and ileum, in this case, of male Long Evens rats, and changes in biochemical composition of intestinal tissues at early stages of growth. The aim of this work was to establish a metabolic profile of normal jejunum and ileum with a view to subsequently evaluating the effects of diet, stress, gut microbiota, and diseases on the metabolic compositions of these tissues.
Experimental Methods Sample Details. All animal studies were carried out under appropriate Swiss national guidelines. Male Long Evens rats (n ) 7) age 3-4 months weighing approximately 400 g were sacrificed by decapitation. Intestines were removed onto icecold glass dishes and flushed with phosphate buffer saline. Cross sections of jejunum and ileum were cut and immediately snap frozen in liquid nitrogen and stored at -80 °C until required for NMR spectroscopic analysis. The same tissues were
research articles also analyzed using a proteomic technique and this has been published elsewhere.34 To investigate the changes of the biochemical profiles of intestinal tissues during the early stage of growth, a separate experiment was carried out. Rats (n ) 6) were sacrificed at 15 and 36 days old and jejunum and ileum were removed from each animal. The criteria for sampling jejunum and ileum were as follows: the first 12 cm of intestine from stomach was considered as duodenum and the rest of intestine was divided into three sections, the first 2/3 were designated as jejunum and remaining 1/3 as ileum. Samples were taken in the middle of each section to avoid variation due to possible differences in the length of sections. 1 H HRMAS NMR Spectroscopy. Samples of jejunum and ileum tissue, each approximately 15 mg, were soaked in D2O and were packed into separate 4 mm diameter zirconia rotors with spherical inserts and Kel-F caps. All NMR experiments were carried out on a Bruker DRX-600 spectrometer (Bruker Biospin, Rheinstetten, Germany), at 283 K, operating at a 1H frequency of 600.13 MHz. Samples were spun at 5 kHz at the magic angle. A total of 15 min was allowed for temperature equilibration before NMR acquisition. A standard Bruker highresolution MAS probe with a magic-angle gradient was employed and the 90° pulse length was adjusted individually for each sample, having a value between 9.6 and 10 µs. A total of 128 transients were collected into 16 k data points for each spectrum with a spectral width of 20 ppm and a recycle delay of 2.0 s. Two 1H NMR spectra were acquired for each tissue, a standard one-dimensional pulse sequence, using the first increment of the noesy pulse sequence to achieve water presaturation (90-t1-90-tm-90-acq)35 and a Carr-Purcell-Meiboom-Gill (CPMG) [90-(τ-180-τ)n-acq] pulse sequence to enhance the contribution of low molecular weight metabolites.36 For the standard one-dimensional experiment, the interpulse delay t1 was 3 µs and the mixing time tm was 100 ms. A weak irradiation was applied at the water resonance during both the mixing time and the recycle delay. For the CPMG experiment, a spin-spin relaxation delay, 2 nτ, of 225 ms was used for all samples and water signal irradiation was applied during the recycle delay. For assignment purposes, two-dimensional (2D) 1H-1H COrrelation SpectroscopY (COSY) and TOtal Correlation SpectroscopY (TOCSY) NMR spectra were also acquired for selected jejunum and ileum tissues. In both cases, 48 transients per increment and 256 increments were collected into 2k data points. The spectral width in both dimensions was 10 ppm. The TOCSY NMR spectra were acquired using the MLEV-1737 spin-lock scheme for 1H-1H transfers with a spin-lock power of 6 kHz. The COSY spectra were recorded using gradient selection. In both types of 2D NMR experiments, the water signal was irradiated with a weak pulse (∼50 Hz) during the recycle delay and for both experiments; the data were zerofilled to 2k data points in the evolution dimension. Prior to Fourier transformation, an unshifted sine-bell and a shifted sine-bell apodization function were applied to the free induction decays of the COSY and TOCSY spectra, respectively. Data Analysis. For all 1D spectra, free induction decays were multiplied by an exponential function with a 0.3 Hz linebroadening factor prior to Fourier transformation and were phased and baseline-corrected using XWINNMR 3.5 (Bruker). The spectra over the range δ 0.6-10.0 were digitized using Matlab script developed in-house (Dr. O. Cloarec, Imperial College London). The region δ 4.7-5.2 was removed to avoid Journal of Proteome Research • Vol. 4, No. 4, 2005 1325
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Figure 1. 600 MHz HRMAS 1H NMR spectra of intact rat (A) jejunum and (B) ileum acquired using the CPMG pulse sequence. The aromatic region of jejunum (A) was magnified 4 times and that of ileum (B) was magnified 8 times compared with corresponding aliphatic region for the purpose of clarity. Peaks are numbered and the assignments are given in Table 1. The rotation rate was 5 kHz.
the effects of imperfect water suppression. Ethanol resonances (δ 1.17-1.23 and δ 3.62-3.67) were also removed due to volatile nature of this compound. Normalization to the sum of the spectrum respectively was carried out on the data prior to pattern recognition analyses. Principal Component Analysis (PCA) was carried out on mean centered data with the software Simca-P 10.0 (Umetrics, Umeå, Sweden). Data were visualized in the form of the PC scores plots and loadings plots. Each coordinate on the scores plot represents an individual sample and each coordinate on the loadings plots represents one NMR spectral data points relating to metabolites. Thus, the loadings plots provide information on spectral regions responsible for the position of coordinates or clusters of samples in the corresponding scores plots.
Results and Discussion 1 H HRMAS NMR spectra of intestinal tissues: Typical 1H HRMAS NMR spectra of intact rat ileum and jejunum tissues, obtained from 3 to 4 months old rats, acquired using the CPMG NMR pulse sequence are shown in Figure 1. Spectra acquired using the standard one-dimensional sequence were similar, but showed also broad components from macromolecules that are attenuated by the use of the CPMG sequence. The onedimensional spectra are often referred to as unedited 1H NMR spectra since this experiment allows all visible protons signals to be detected including signals from both small metabolites and large macromolecules and is generally representative of total biochemical composition. The CPMG spectra are referred to as T2-edited spectra and in these spectra, the lipid signals are substantially attenuated and the relative intensities of those resonances from the small molecules such as amino acids are clearly enhanced. The NMR resonances in these 1D spectra can only be assigned tentatively by comparing with the literature data. To make unambiguous assignment, it is necessary to employ two-dimensional (2D) 1H-1H COSY (not shown) and TOCSY (Figure 2) NMR spectra of intact tissue. The COSY and TOCSY are standard NMR methods which have been
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Figure 2. 1H-1H total correlation (TOCSY) spectra of ileum acquired at 600 MHz at a spinning rate of 5 kHz. The numbering system is the same as used in Figure 1 and Table 1.
proven useful for identification of metabolites. The cross-peaks in COSY spectra indicate pairs of directly coupled protons, whereas the cross-peaks in TOCSY spectra reveal protonproton connectivity up to five or six bonds, and hence provide
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NMR Spectroscopy of Rat Intestine Development Table 1. 1H NMR Chemical Shift Assignments of Small Intestine Tissues code 1
2
3
metabolites
group
Leucine
R-CH β-CH2 γ-CH δ-CH3 δ-CH3 R-CH β-CH γ-CH2 γ-CH3 δ-CH3 R-CH β-CH γ-CH3 γ-CH3 R-CH2 β-CH3 R-CH β-CH3 R-CH β-CH γ-CH3 R-CH β-CH3 R-CH β-CH2 γ-CH2 δ-CH2 -CH2 R-CH β-CH2 γ-CH2 δ-CH2 R-CH β-CH2 γ-CH2 δ-CH2 R-CH β-CH2 γ-CH2 R-CH β-CH2 γ-CH2 δ-CH3
Iso-leucine
Valine
4
Ethanol
5
Lactate
6
Threonine
7
Alanine
8
Lysine
9
Arginine
10
Proline
11
Glutamate
12
Methionine
δ 1H (ppm) (multiplicitiy) 3.72 (t) 1.96, 1.63 (m) 1.69 (m) 0.91 (d) 0.94 (d) 3.65 (d) 1.95 (m) 1.25, 1.45 (m) 0.99 (d) 1.02 (d) 3.6 (d) 2.26 (m) 0.98 (d) 1.04(d) 1.18 (t) 3.65(q) 4.11 (q) 1.32 (d) 3.59 (d) 4.25 (m) 1.32 (d) 3.77 (q) 1.47 (d) 3.77 (t) 1.89 (m) 1.72 (m) 1.47 (m) 3.01 (t) 3.76 (t) 1.89 (m) 1.63 (m) 3.23 (t) 4.11 (t) 2.02 (m), 2.33 (m) 2.00 (m) 3.34 (t) 3.75 (m) 2.08 (m) 2.34 (m) 3.78 (m) 2.14 (m) 2.60 (dd) 2.13 (s)
additional conformation. By combination of the 1H-1H COSY and TOCSY methods, the resonances of lysine and arginine, proline and glutamate were clearly separated and unambiguously assigned, which are otherwise overlapped in the 1D spectra and can only be tentatively assigned. Furthermore, cross-peaks from the R- and β-CH2 groups of choline, and the R- and β-protons of serine were also easily identified in the two-dimensional 1H-1H TOCSY spectrum (Figure 2). The resonances assigned based on the 1D and 2D spectra and literature values35 are tabulated in Table 1 together with the corresponding 1H NMR chemical shifts and signal multiplicities. By employing HRMAS NMR spectroscopic techniques, our studies on both the jejunum and ileum in the rodent small intestine revealed that the molecular species there include a number of amino acids, carboxylic acids such as acetate and lactate, pyrimidines including cytosine, isocytosine, uracil, and membrane component metabolites such as glycerophosphocholine (GPC). In addition, choline, lipids, creatine and ethanol were also identified. Similarities and differences were found between human gastric mucosa and rat intestine tissues. For example, amino acids and fatty acids were commonly found in human gastric mucosa and rat intestine tissues, whereas more membrane component metabolites were found in human
code
metabolites
group
13
Glutamine
14
Aspartic acid
15
Asparagine
16
Ethanolamine
17
Choline
18
GPC
19
Taurine
20 21
Glycine Creatine
22 23
Acetate Uracil
24
Isocytosine
25
Cytosine
26
Tyrosine
27
Phenylalanine
28
Serine
29
Lipid
R-CH β-CH2 γ-CH2 R-CH β-CH2 R-CH β-CH2 NH9CH2 HO9CH2 N-(CH3)3 R-CH2 β-CH2 N-(CH3)3 R-CH2 β-CH2 R′-CH2 β′-CH γ′-CH2 N-CH2 S-CH2 R-CH N-CH3 CH2 CH3 CH CH CH CH CH CH CH CH 2,6-CH 3,5-CH 4-CH R-CH β-CH2 CH3 (CH2)n CH2CdC CH2-CdO )C-CH2-C) -CHdCH-
δ 1H (ppm) (multiplicitiy) 3.77 (m) 2.15 (m) 2.44(m) 3.89 (m) 2.79 (m); 2.82(m) 3.99 (m) 2.86 (m); 2.94(m) 3.13 (t) 3.83 (t) 3.20 (s) 4.05 (t) 3.51(t) 3.22 (s) 4.32 (t) 3.68 (t) 3.60 (dd) 3.89 (m) 3.72 (dd) 3.26 (t) 3.40 (t) 3.55 (s) 3.03(s) 3.92 (s) 1.91(s) 5.78 (d) 7.52(d) 6.03(d) 7.82(d) 5.86(d) 7.84(d) 7.16 (dd) 6.87(dd) 7.40 (m) 7.33 (m) 7.35 (m) 3.84 (m) 3.96 (m) 0.89 (m) 1.27 (m) 1.8 (m) 2.0 (m) 2.78 (m) 5.3 (m)
gastric mucosa.31 In addition, a singlet resonance at δ3.7, which was not assigned, appeared to be unique for human gastric mucosa.31 The finding of abundant amino acids and lipids in the jejunum and ileum is not surprising, as these represent some of the main nutrients absorbed by the intestine. For example, it is also well documented that glutamine is important in maintaining the function and integrity of the intestine.38 Glutamine is required by villous enterocytes to support enterocyte metabolism, structural integrity and the function of the small intestine of dogs and rodents.39,40 Wu et al. reported that the addition of 1% glutamine to the diet prevented villous atrophy in the jejunum on the seventh day after weaning.41 Furthermore, several proteins, such as the actin family involving contractility of muscle, and calponin-H1 involved in regulation and modulation of smooth muscle contraction, were detected in intestine tissues.34 Cellular retinoic acid-binding protein which belongs to the fatty acid-binding protein family was also detected in intestine tissues, consistent with the fatty acids found in the NMR profiles of jejunum and ileum.34 Absorption of nutrients is the most important function of the small intestine. However, measurement of such absorption can be a complicated procedure using conventional biochemiJournal of Proteome Research • Vol. 4, No. 4, 2005 1327
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Figure 3. PC1 vs PC2 scores plot (top) and corresponding loadings plot (bottom) derived from the 1H HRMAS NMR spectra of jejunum (9) and ileum (0) of 3-4 months old rats.
cal techniques. The 1H HRMAS NMR spectroscopic approach demonstrated here offers an alternative, which enables simultaneous detection of a range of biochemical components in intestine using a typical biopsy size tissue sample (∼15 mg). In addition, this approach is nondestructive and samples can be retrieved after NMR data acquisition for further analysis. Differences between Jejunum and Ileum. From visual comparison, the 1H HRMAS NMR spectra of jejunum and ileum were similar in terms of their biochemical composition. Therefore, PCA was carried out on the NMR of the T2-edited NMR spectra data of these tissues to further investigate biochemical differences between jejunum and ileum tissues (Figure 3 top). Similar results were obtained from the comparison of the standard 1D NMR experiments. Three principal components were calculated, with the first two PCs explaining 95.2% of the total variances within the data. PC1 was dominated by an outlying, sample number 7, which contained relatively higher levels of choline and GPC than the other samples. Separation of jejunum from ileum was observed in PC2 except for the sample number 2 from ileum, which appeared to be clustered with jejunum. The spectral regions that contributed most to the difference between ileum and jejunum are shown in the corresponding loading plot (Figure 3 bottom). The metabolites exerting the greatest influences on this separation were GPC, lactate, creatine, choline, acetate, lipids, and a range of amino acids including leucine, isoleucine, valine, methionine, and taurine. Although some of differences between these two tissues profiles were difficult to distinguish by eye the PC analysis indicated that the jejunum contained relatively higher levels of lipids, lactate, creatinine, choline, GPC, and lower levels of acetate and amino acids than the ileum. One of the function of the ileum is to facilitate the active absorption of bile acids and taurine conjugated bile acids,42 hence possibly accounts for the high level of taurine found in ileum tissues in the current study. In addition, a marker protein, gastrotropin, was found to be specific to ileum tissues in proteomic studies 1328
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Figure 4. PC1 vs PC2 scores plot (top) and corresponding loadings plot (bottom) derived from the 1H HRMAS NMR spectra of jejunum obtained from 15 (0), 36 (b) days old rats, and 3-4 months old rats (O).
of the same tissues.34 Gastrotropin is a major intracellular transporter of bile acids in enterocytes and is involved in the transport of bile acids from the brush border membrane to the basolateral pole of the ileocyte.34,43 Previous investigation of storage location for digested dietary fats in intestine suggested that fat is retained within the jejunal tissues,44 which is consistent with our observation of relatively higher level of lipids in jejunum tissues compared with the ileum. GPC is a membrane intermediate in membrane metabolism pathways; however, the cause of higher concentrations of GPC observed in jejunum compared to ileum is unclear. Two jejunum tissues appeared well separated from the other five-jejunum tissues due to these two samples containing relatively higher levels of lipids as confirmed by inspection of the NMR spectra. Although jejunum and ileum are not markedly different in their gross anatomical structure, they are functionally and histologically distinct and are colonized by different bacterial complements.7 The results shown here illustrate that jejunum and ileum have consistent differences in terms of their biochemical constitutes. This observation suggests that care should be taken to maintain accuracy of localization when sampling from the intestine. Age Dependence of Intestinal Tissues. PCA was also carried out on the T2-edited NMR spectral data of intestinal tissues including jejunum and ileum obtained from 15 days, 36 days old rats to investigate changes in intestine biochemical composition during early growth (Figure 4). Clear separation between jejunum obtained from 15 days, 36 days, and 3-4 months old rats were observed. Inspection of the corresponding loadings suggested that relative higher concentrations of GPC, amino acids, such as leucine, isoleucine, alanine, valine, glycine, arginine, proline, glutamine, methionine, and lysine were found in jejunum obtained from 15 days old rats whereas relatively higher concentrations of lipids, lactate, glucose,
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NMR Spectroscopy of Rat Intestine Development
creatinine, and taurine were found in the older rats. This observation suggested that the amino acids absorption declined with age, which is consistent with previous reports on agerelated amino acid uptake.45,46 In contrast to jejunum, ileum obtained from 36 days old and 3-4 months old rats appeared to be scattered due to variation of lipids presented in tissues whereas those obtained from 15 days old rats are clustered closely. It is known that development of intestine is highly dependent on the microbiota12-14 and that ileum contains high numbers of bacteria compared to jejunum.8 Recently, study of impact of gut microbiota on fat storage suggested that introduction of gut microbiota into adult germ free-mice produced a rapid increase in body fat due to additional energy from microbiotia degradation of complex polysaccharides.47 However, no direct link between bacterial colonization and lipid storage in intestine tissues has been established. In summary, biochemical components of jejunum and ileum in rats were characterized using an nondestructive 1H highresolution magic-angle-spinning NMR spectroscopy and a comprehensive 1H NMR resonance assignment was made by the assistance of 1H-1H TOCSY and COSY NMR spectra. Jejunum and ileum are metabolically similar in composition and are mainly comprised of amino acids, lipids, GPC, choline, creatine, ethanol, cytosine, isocytosine, uracil, acetate, and lactate. PCA of the NMR data facilitated the differentiation of the two tissues and indicated that the jejunum contained higher levels of lipids, lactate, GPC, choline, and creatinine, whereas the ileum contained higher levels of acetate and amino acids. Age dependence of biochemical composition of intestinal tissues was also studied by comparing the tissues obtained from rats at 15 and 36 days old, which were found to differ significantly in biochemical compositions. This work demonstrated that 1H HRMAS NMR spectroscopy of intestinal tissue produced robust metabolic signatures for jejunum and ileum tissue and was able to differentiate the metabolic signature for each tissue according to age. This work will serve as a reference with which to compare changes in intestinal composition in response to diet, age, disease, and gut microbiota. Abbreviations: CPMG, Carr-Purcell-Meiboom-Gill; GPC, glycerophosphocholine; HRMAS, high-resolution magic angle spinning; NMR, nuclear magnetic resonance; PCA, principal component analysis; COSY, 1H-1H correlation spectroscopy; TOCSY, 1 H-1H total correlation spectroscopy.
Acknowledgment. Nestec S.A., Switzerland is acknowledged for the funding of Dr. Y. Wang. References (1) Xu, J.; Bjursell, M. K.; Himrod, J.; Deng, S.; Carmichael, L. K.; Chiang, H. C.; Hooper, L. V.; Gordon, J. I. Science 2003, 299, 20742076. (2) Bragg, L. E.; Thompson, J. S.; Rikkers, L. F. Nutrition 1991, 7, 237243. (3) Ferraris, R. P.; Hsiao, J.; Hernandez, R.; Hirayama, B. Am. J. Physiol. 1993, 264, G285-G293. (4) Kiliaan, A. J.; Saunders, P. R.; Bijlsma, P. B.; Berin, M. C.; Taminiau, J. A.; Groot, J. A.; Perdue, M. H. Am. J. Physiol.-Gastr. L 1998, 38, G1037-G1044. (5) Dou, Y. L.; Gregersen, S.; Zhao, J. B.; Zhuang, F. Y.; Gregersen, H. Digest Dis. Sci. 2002, 47, 1158-1168. (6) Barada, K. A.; Kafrouni, M. I.; Khoury, C. I.; Saade, N. E.; Mourad, F. H.; Szabo, S. S.; Nassar, C. F. Life Sc. 2001, 69, 3121-3131. (7) Xu, J.; Gordon, J. I. Proc. Natl. Acad. Sci. USA 2003, 100, 1045210459. (8) Simon, G. L.; Gorbach, S. L. Gastroenterology 1984, 86, 174-193. (9) Cebra, J. J. Am. J. Clin. Nutr. 1999, 69, 1046S-1051S.
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