The complex links between dietary
phytochemicals and human health
deciphered by metabolomics
: opportunities and challenges
Mol. Nutr. Food Res. 2009, 53, 1303 – 1315 Am J Clin Nutr 2005;82:497–503
Introduction
• A large variety of phytochemicals(植化素)
commonly consumed with the human diet,
influence health and may contribute to the
prevention of diseases.
• However, it is still difficult to make nutritional
recommendations for these bioactive
compounds
• Current studies of phytochemicals are generally focused on specific compounds and their effects on a limited number of markers
• New approaches are needed to take into
account both the diversity of phytochemicals found in the diet and the complexity of their biological effects
• Metabolomics studies illustrate the potential of such a global approach to explore the complex relationships linking phytochemical intake and metabolism and health.
• A regular consumption of fruits, vegetables
and whole grains is associated with reduced
risks of developing chronic diseases such as
cancer and cardiovascular diseases
• More than 5000 individual phytochemicals
have been identified in food and beverages
carbohydrates and lipids
They can be classified into six major groups
called isoprenoids
萜類 :萜類是廣泛 布在自然界 並且在高等植物中含量很高,真 菌類產生許多萜類；海中生物
alkaloid」泛指一 含有 氮的鹼基。 雖然大部份 的生物鹼對於人體有毒， 但也有些能入藥。主要 有鎮痛或麻醉的作用， 以嗎啡及可待因的作用 尤其顯著
生物鹼
青花菜中的硫配醣體有助於防癌和預防心血管疾病
• A large variety of biological effects and
mechanisms of action have been described depending on their chemical structures
• Plant sterols, reduce LDL-cholesterol levels in humans by interfering with cholesterol intestinal absorption
• Soy isoflavones: estrogenic or anti-estrogenic properties reduced risk of some hormone-dependent diseases (tested in vitro)
• it is still difficult to make nutritional
recommendations for phytochemicals.
• Beyond elucidation of their mechanisms of
action, such recommendations should be
largely based on randomised control trials
and epidemiological studies
• However, most often the number of
intervention studies is still too limited to
draw definitive conclusions on the effects
of phytochemicals on health.
• Short-term studies
• Foods rich in phytochemicals, rather than
pure phytochemicals
• The limited number of phytochemicals is
far from representing the wide diversity of
compounds consumed with the diet.
• New tools and approaches are needed to
take into account the following two levels
of complexity:
• (i) the diversity of phytochemicals in the
human diet
• (ii) the complexity of their biological effects.
• The metabolome can be divided into
several fractions:
• the endogenous: cell, a tissue or an
organism
• the microbial metabolome
• food metabolome
• the xenometabolome ( characterization of
drug metabolites ) which includes all
foreign metabolites derived from drugs,
pollutants and dietary compounds
modulate cell metabolism,
Health and disease risk
Endogenous metabolites and exogenous phytochemical metabolites form a signature characteristic of the intake of a given phytochemical.
This signature contains detailed information on phytochemical intake and on the effects of these phytochemicals on host metabolism.
Genome
Measuring dietary intake of
phytochemicals: Current limitations
• Dietary records and food composition
tables for phytochemicals are required to
estimate intake
• Dietary intakes are based on food
frequency questionnaires or multiple 24 h
recalls.
• Limitations: the accuracy of such
questionnaires and self-reports remains
uncertain
Food composition tables for
phytochemicals
• US Department of Agriculture related to the levels of isoflavones, flavonoids, procyanidins or carotenoids in selected foods (http://www.nal.usda.gov/fnic/foodcomp/Data/isoflav/isoflav.html,
http://www.nal.usda.- gov/fnic/foodcomp/,http://www.nal.usda.gov/fnic/foodcomp/Data/PA/PA.html)
• Phenol-Explorer database for all polyphenols including phenolic acids
• VENUS database related to the levels of phytoestrogens in plant foods, databases dedicated to the glucosinolates in cruciferous vegetables or phytosterols in various
foods
• These databases are still incomplete in regard to the considerable diversity of phytochemicals in food plants.
• Various parameters such as genetic,
environmental factors (including growing location or agricultural practices)and food
processing and storage have a profound effect on the levels of phytochemicals in food.
• These varieties are often not distinguished either in the food composition tables or in dietary
records, making the estimation of the phytochemical intake less accurate. Solution:
• The direct estimation of phytochemicals in human urine or plasma allows to partly
circumvent these limitations.
Biomarkers of phytochemical intake
• Phytochemicals, once absorbed, are found in the
systemic circulation, either unchanged or in the form of various metabolites and are eventually excreted in urine.
• Carotenoids are absorbed through the gut and some of them (provitamin A carotenoids) are cleaved in the
intestine and in the liver to form vitamin A and other breakdown products.
• Concentrations of these metabolites in urine or plasma usually reflect the amount of phytochemicals ingested
• Plasmatic or urinary isoflavones( 大豆異黃
酮(), lignans(植物雌激素) and
carotenoids(類胡蘿蔔素) have effectively
been used as biomarkers of intake in
various epidemiological studies to look for
associations with disease or disease risk
The selection of candidate
biomarkers
• Previous knowledge on the metabolism and pharmacokinetics of the phytochemicals of interest
• Various factors which may influence their inter-individual variations in absorption, metabolism and excretion
• The factors which affect the reliability of such candidate markers of intake
phytochemical lifetime in the body
• Reflect long-term intake: carotenoids
• short-term intake: chlorogenic (coffee;綠原酸 ) and catechins (tea;兒茶素 )
• Quickly eliminated within less than a day, can still be used as biomarkers of intake due to the very regular consumption of their major food sources by some individuals
Second factor influencing the
reliability of biomarkers
• the composition of the diet and the food
matrix may interfere with the absorption of
some phytochemicals in the gut
• Plasma concentrations of lycopene are higher after consumption of a tomato puree rather than fresh raw tomatoes
• Carotenoid bioavailability was shown to depend on the lipid content of the diet
• Bioavailability of green tea catechins was also significantly enhanced when consumed in fasting conditions rather than with a meal
• Plasma concentrations may better reflect
tissular exposure than intake
Absorption and metabolism of
phytochemicals can also differ widely
between individuals
• Genetic polymorphism, physiological state
and gut microbiota
The nature of the biofluid from
which the phytochemical
• Blood
• Urine
• Saliva
• hormones such as 17-OH progesterone, testosterone, estradiol, and free cortisol as a biomarker of plasma arachidonic acid
• Fecal water : microfolra
• More invasive :
cerebrospinal fluid, liver, gut, or muscle biopsy specimens• Many phytochemicals with short half-lives show large variations of concentrations in the plasma over one day
• Lesser variations are expected in urine due to their accumulation over several hours.
• Spot urine samples vs 24-h urine samples
• Good correlations were observed between most polyphenols and the consumption of their major food sources in both types of urine samples
• Quantify the set of selected compounds in
urine or plasma in a targeted approach
• Alternatively, a metabolomics
fingerprinting approach could be used to
identify new and unexpected candidate
biomarkers of intake for phytochemicals
Metabolomics and biomarker
discovery
• NMR and MS
• statistical tools: unsupervise vs supervise
unsupervise (no prior knowledge of sample classes)
• principal component analysis (PCA) or hierarchical clustering analysis (HCA).
supervise
• Partial Least Square Discriminate Analysis (PLS-DA)
• Visualize metabolic differences between predefined sample classes
Exact mass and mass fragments
KEGG Ligand Database (http://www.genome.jp/kegg)
contain a limited numberof phytochemicals and virtually no conjugated metabolites
PubChem Project (http://pubchem.ncbi.nlm.nih.gov)
More than 12 million compounds, covers a wide range of phytochemicals as found in plants, but few of their metabolites. Ex 11/23 metabolites of quercetin detected in human plasma or urine after ingestion of onions
Human Metabolome Data-Base (http://www.hmdb.ca) Metabolome Japan (http://www.metabolome.jp)
METLIN database (http://metlin.scripps.edu/)
Limits of the current methodologies
• The lack of well established and standardized
methods or procedures
• Insufficient coverage of the human metabolome
by current analytical procedures
• Insufficient data exploitation or data overfitting,
incomplete identification of the metabolites
• Lack of bioinformatic tools to interpret changes
in metabolomics fingerprints
• Lack of standards for absolute quantification
Metabolomics and phytochemical
intake
• A limited number of phytochemical metabolites have so far been studied as markers of
phytochemical or plant food intake
• “one metabolite–one food”
• 100–300 phytochemicals are commonly described in any given plant foods as reported in the Dr.
Duke's Phytochemical and Ethnobotanical
Database (www.ars-grin.gov/duke/plants.html).
• LC-ToF-MS:
• GC-MS:
different phenolic compounds, ferulic acid,
sinapic acid or lignins (urine)
• Each fingerprint comprised a large number
of characteristic phenolic metabolites,
providing new information of the
metabolism of these compounds.
Example
(Am. J. Clin. Nutr. 2007, 86,1687–1693).
• In a controlled intervention study comparing a 2 day-consumption of a low-phytochemical diet and 2 day-consumption of the same diet
supplemented with fruit and vegetable drinks,
• MS and NMR :urine
• Phytochemicals in fruits and vegetables
influence the food metabolome which comprises metabolites which could be used as markers of phytochemical intake.
limited to a few markers and the
significance of the results depends on the value of the hypothesis
GWAS
Candidate
• Identification of phytochemical metabolites in
urine or plasma fingerprints is necessary to
determine the parent phytochemical using
them as biomarkers of intake of the
corresponding phytochemical
• This identification is still difficult due to the lack
of comprehensive databases for
phytochemical metabolites and the lack of
appropriate standards
• Human genome project---> complete human
sequence
• RFLP, STRP 5M SNP commercial platform
• 美國塔弗茨大學營養與基因組研究室的主任奧多維斯
Jose M. Ordovas 說： 相較之下，基因定序的工作
容易多了 他認為定序只需要處理四種成份 A C T
和G ， 而在代謝體學中，你卻得以不同的技術，利用
不同的方式來測定成份，而這些成份有上千種
• 科學家認為基因組學要有真正的進展，必須先建立一個相 當於人類基因組的代謝組 metabonome ：人體細胞和組 織中所有代謝物的資料 但是奧多維斯說，這個領域缺乏 協調和經費 他估計，代謝組分析工作可能需要動員50萬 名以上的人力，才能夠完成這項任務
出自科學人2005年4月號
Metabolomics and biological effects of
phytochemicals
• Due to their high diversity, phytochemicals can affect a wide array of physiological functions and metabolic pathways.
• Evaluate the effects of phytochemicals in short- term clinical trials may fail to accurately describe or predict health effects of phytochemicals.
• Several long-term intervention studies have
failed to show any protective health effects (total mortality) upon supplementation with some
antioxidant vitamins
• Global metabolic changes resulting from a phytochemical intervention are characterized with no restriction to an a priori selected
metabolic pathway, as most commonly done up to now.
• The generation of novel hypotheses on
unexpected modes of action of phytochemicals and to the discovery of new markers of effects able to characterize the subtle metabolic
changes induced in nutritional interventions (Data driven)
Examples
• The consumption of green tea by healthy
volunteers was shown to result in a significant increase in the levels of several citric acid cycle intermediates such as citrate, pyruvate and
oxaloacetate, also suggesting an effect of tea flavanols on oxidative energy metabolism
• The consumption of chamomile tea by healthy subjects was shown to decrease the urinary excretion of creatinine and to increase that of hippurate and glycine, indicating a possible
effect of chamomileon gut microflora metabolism
Limitations
• Targeted methods rather than open
fingerprinting approach in which a
selection of about 100 to 200 metabolites
a priori known as most abundant in the
samples of interest.
• But still leave unexplored, the major
fraction of the human metabolome.
Interpretation of the metabolic changes
• It is still difficult to interpret an increase or
a decrease in the concentrations of a
metabolite in a given context.
• New bioinformatic tools like NuGOwiki
(http://www.nugowiki.org/index.php/Main_
Page)
• HMDB(http://www.hmdb.ca/) will
contribute to provide such information
Conclusion
• Mammals have also evolved alongside plants, depending on some of them as staple food.
• Mammals have thus been exposed to a variety of phytochemicals for millions of years.
• The challenge today is to interpret the complex relationships between phytochemicals present in the human diet and health taking into account
both the diversity of their chemical structures and the complexity of their metabolic effects.
