Using Metabolomics To Better Understand Functional Foods

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Dayan B. Goodnowe1, Yasuyo Yamazaki1, Bernhard H. J. Juurlink2

1Phenomenome Discoveries Inc. 204-407 Downey Road, Saskatoon, SK, S7N 4L8, Canada; 2Department of Anatomy and Cell Biology, College of Medicine, University of Saskatchewan, Saskatoon, SK, S7N 5E5, Canada

Abstract: Decreasing hypertension and inflammation are important human health objectives. Functional foods are being developed to provide an alternative treatment for mild to moderate symptoms as well as for alternatives to pharmaceuticals for long-term health management. Broccoli has been shown to decrease oxidative stress and associated inflammation and hypertension in stroke-prone spontaneously hypertensive rats. This presentation will focus on how comprehensive metabolomics can be used to understand the activity of broccoli in this animal model of hypertension.

Key words: stroke-prone spontaneously hypertensive rat; broccoli sprout.

Metabolomics, the global analysis of small molecules, or metabolites, in biological samples, is the final link in the systems biology chain. To study what genes are expressed under a given set of conditions one measures the change in the concentration and composition of gene transcripts; and, to study what transcripts get translated, one measures the changes in protein content and concentration. It therefore follows that to study how the actual proteins in a system are functioning, one must measure the changes in metabolites content and concentration. The advent of genomics and genechip technology gave scientists the ability to monitor and quantitate the changes in the expression of every gene within a genome. Proteomic technologies have made available valuable functional information and have provided the ability to monitor gene products. In the systems biology picture, however, humans truly are more than the sum of our parts; we are more than our genes. To monitor the biology of a system, one must be able to measure

the effects of the environment on gene expression. Because an observed phenotype is the summation of gene expression, resultant protein activity, and environmental influences, metabolomics is required to supply the last piece of the puzzle - the quantifiable measurement of the interaction of genetics and environment. Metabolomics offers measurement of phenotype. The changes observed in the metabolome directly indicate what system changes (e.g. genes or drugs) affect the function of which pathways. As well, the exact point(s) in the pathway affected can be determined. Although proteins involved in these pathways may be useful drug or pesticide targets, it is their activity - not their amount - that is important. This is why the correlation between activity and protein concentration is often poor. Of the omics technologies, only metabolomics consistently provides strong correlations between concentration and observed phenotype.

No attention to human health is complete without recognizing the important role that diet and lifestyle play in wellness and disease prevention. It has been estimated that 30 - 40 percent of all cancers can be prevented by lifestyle and dietary measures alone. Since consumption of large quantities of fruit and vegetables is associated with a striking reduction in the risk of developing a variety of inflammations, it is of interest that a number of dietary plants contain substantial quantities of compounds that regulate mammalian enzymes of xenobiotic metabolism. Thus, dietary plants belonging to the family Cruciferae and genus Brassoca (e.g., broccoli and cauliflower) contain substantial quantities of sulforaphane (4-methylsulfinylbutyl isothiocyanate) which is the most potent phase 2 protein inducer found in our food sources (Fahey et al., 1997). A major action of phase 2 proteins, mainly enzymes, is to inactivate electrophiles and strong oxidants (Prestera et al., 1993); hence, there has been a research interest in dietary phase 2 protein inducers as a means to prevent cancers (Talalay and Fahey, 2001) .

The animal model chosen was the male and female stroke-prone spontaneously hypertensive rats (SHPsp) (Wu et al., 2004). This strain develops severe hypertension (Nagaoka et al., 1976) that is related to increased peroxynitrile formation (Ma et al., 2004) and develops inflammatory changes in blood vessels (Liu et al., 1994, 1996). We chose broccoli sprouts as the source of phase 2 protein inducers. We put stroke-prone spontaneouly hypertensive rats (n = 4-5 rats per group) on a special diet of AIN-93 (Reeves et al., 1993) plus 200mg/day broccoli sprouts for a period of 4 months and during this period measured their blood pressure and looked at the state of inflammation and oxidative stress in different organs compared to controls which were on AIN-93 diet only. The mentioned diet controls blood pressure and ameliorates inflammatory changes. At the end of study, anesthetized animals were perfused with buffered saline, and kidneys and plasma were removed and frozen in liquid nitrogen. The frozen tissues were ground in the frozen state, and homogenized tissue powders were used for non-targeted FTMS analysis.

A total of 68 samples (duplicate analysis of control diet SHPsp liver and plasma, male and female n = 4; duplicate analysis of broccoli fed SHPsp liver and plasma, n = 4 male, n = 5 female) were processed via a proprietary extraction method optimized to separate the metabolites into multiple extracts based upon their polarity and acid/base chemistry as shown in Figure 1. Extracts were analyzed by direct injection Fourier Transform Ion Cyclotron Resonance mass spectrometry (FT-ICR-MS). This is currently the highest resolution mass spec technology available, and is the only method that provides the degree of resolution and mass accuracy required to reliably separate metabolites in complex samples. The resulting data set can easily be visualized using DISCOVAmetrics™, a bioinformatics platform that allows multiple samples to be easily compared and mined for both known and novel metabolites.

Figure 1. Comprehensive metabolome analysis technology

To evaluate internal reproducibility, each sample was extracted and analyzed in duplicate. The correlations between the two replicates are plotted in Figure 2. The R-squared values for the total average was 0.96.

Figure 2. QAQC reproducibility

Global statistics were performed using all samples, and setting each tissue and gender (n of 16 or 18) equal to one variable, for total of 4 variables. Following log(2) normalization and significance testing (^-values < 0.01). As shown in Figure 3, each tissue was completely separated.

Figure 3. PCA

To evaluate the relative difference between the two treatments (broccoli fed vs. control) in liver and plasma, all male samples were normalized to male control average of each detected metabolite and all female samples were normalized to female control average of each detected metabolite. Hierarchical Clustering Analysis (HCA) plots were generated (see Figure 4).

Figure 4. HCA plots

Interestingly, in Figure 4A plasma samples, females exhibit a greater effect of broccoli feeding, while in liver samples at Figure 4B, males and females show different responses to broccoli feeding. Tables 1 and 2 show the number of the significant metabolite changes (_p-values < 0.05) between broccoli fed samples vs. control samples in plasma and liver, respectively. According to Table 1, females show a greater effect of broccoli feeding as seen in the HCA result in Figure 4A. In Table 2, a total of 108 metabolites were down-regulated in female liver samples. This phenomenon is totally different from male samples. Focusing on detailed metabolites in plasma, 30 metabolites were up-regulated, including fatty acids, diacylglycerols and triacylglycerols. Additionally this phenomenon was more observed to be greater in female samples. In male liver samples, mainly fatty acids, diacylglycerols and triacylglycerols were up-regulated. On the other hand in female liver samples, glucuronidated metabolites were down-regulated.

Table 1. Number of metabolites which expressed significantly in plasma samples (p-values < 0.05)_

plasma

plasma

male + female

male

female

> 2.0x

3

4

9

1.2x < 2.0x

52

33

99

total up

74

45

127

0.5x < 0.8x

6

7

5

< 0.5x

2

6

2

total down

15

51

8

total changed

89

96

135

Table 2. Number of metabolites which expressed significantly in liver samples (p-values < 0.05)_

liver

liver

male + female

male

female

> 2.0x

0

4

1

1.2x < 2.0x

16

44

28

total up

20

85

38

0.5x < 0.8x

8

13

83

< 0.5x

0

0

1

total down

36

58

108

total changed

56

143

The data provided in the current study demonstrates the utility of the comprehensive metabolomics analysis provided in DISCOVAmetrics™ technology. Metabolic analysis is a potentially powerful tool that can be used to address both applied and basic research questions. Broccoli feeding clearly elicited different responses in male and female animals. Such analyses will increase our understanding of the complex pharmacological antioxidants and provide new perspectives for understanding human responses.

REFERENCES

Fahey, J. W., Zhang, Y. and Talalay, P., 1997, Broccoli sprouts: An exceptionally rich source of inducers of enzymes that protect against chemical carcinogens, Proc. Natl. Acad. Sci. USA 94:10367-10372.

Liu, Y., Jacobowitz, D. M., Barone, F., McCarron, R., Spatz, M., Feuerstein, G., Hallenbeck, J. M., and Siren, A. L., 1994, Quantitation of perivascular monocytes and macrophages around cerebral blood vessels of hypertensive and aged rats, J. Cereb. Blood Flow Metab. 14:348-352.

Liu, Y., Liu, T., McCarron, R. M., Spatz, M., Feuerstein, G., Hallenbeck, J. M. and Siren, A. L., 1996, Evidence for activation of endothelium and monocytes in hypertensive rats, Am. J. Physiol. 270:2125-2131.

Ma, X.-L., Gao, F., Nelson, A. H., Lopez, B. L., Christopher, T. A., Yue, T.-L. and Barone, F. C., 2001, Oxidative inactivation of nitric oxide and endothelial dysfunction in stroke-prone spontaneous hypertensive rats, J. Pharmacol. Exp. Ther. 298: 879- 885.

Nagaoka, A., Iwatsuka, H., Suzuoki, Z. and Okamoto, K, 1976, Genetic predisposition to stroke in spontaneously hypertensive rats, Am. J. Physiol. 230:1354-1359.

Prestera, T., Holtzclaw, W. D., Zhang, Y. and Talalay, P., 1993, Chemical and molecular regulation of enzymes that detoxify carcinogens, Proc. Natl. Acad. Sci. USA 90:2965-2969. Reeves, P. G., Nielsen, F. H., Fahey, G. C. Jr., 1993, AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939-1951. Talalay, P, and Fahey, J. W., 2001, Phytochemicals from cruciferous plants protect against cancer by modulating carcinogen metabolism, J. Nutr. 131:3027S-3033S.

Wu, L., Ashraf, M. H. N., Facci, M., Wang, R., Paterson, P. G., Ferrie, A. and Juurlink, B. H., 2004, Dietary approach to attenuate oxidative stress, hypertension, and inflammation in the cardiovascular system, Proc. Natl. Acad. Sci. USA 101:7094-7099.

FOOD FACTORS THAT REGULATE INTESTINAL INFLAMMATION: EVALUATION OF THE FACTORS BY USING A COCULTURE SYSTEM

Hideo Satsu and Makoto Shimizu

Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan

Abstract: Immune cells located in the intestinal epithelium interact with intestinal epithelial cells via soluble factors. A new in vitro model using a coculture system was constructed in this study to analyze the interaction between intestinal epithelial cells and macrophage-like cells. Human intestinal epithelial Caco-2 cells were differentiated on semipermeable membranes. Human monocytic THP-1 cells were differentiated to macrophage-like cells and then cocultured on the basal side of the Caco-2 monolayers. Coculturing for 48 hours resulted in a decrease in the transepithelial electrical resistance of the Caco-2 monolayers and an increased release of lactate dehydrogenase from the Caco-2 cells. This suggests that the coculture with THP-1 induced some disruption in the Caco-2 monolayers. A conditioned medium of the THP-1 cells gave similar results. This disruption was significantly suppressed by adding the anti-TNF-a antibody to the medium, suggesting that TNF-a secreted from THP-1 caused damage to the Caco-2 cells. It is also seems that this phenomenon is similar to that observed with inflammatory bowel disease (IBD). The effects of food factors on the cells in this coculture system were examined. The disruption of the Caco-2 cell monolayers was significantly reduced by adding caffeine to the medium which suppressed the secretion of TNF-a from the THP-1 cells. This model will be useful to study the inflammatory damage to cells in the intestinal epithelium such as IBD, and also the protective functions of food factors involved in this process

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