Biomarkers for Food Components With
Health Benefits: Progress and Issues
Carl L. Keen
Department of Nutrition
University of California, Davis
clkeen@ucdavis.edu
Food Forum & the ILSI North American
Project Committee on Flavonoids
Washington, D.C.
June 8th, 2010
Diets rich in plant foods are strongly associated
with a decreased risk for cardiovascular disease.
Is this a true causative relationship?
If yes, what are the factors driving it?
CHD
IHD
Dauchet et al., J Nutr 136: 2588-93, 2006
Fruits, vegetables and coronary heart disease
“ Evidence that fruit and vegetable consumption reduces
the risk of cardiovascular disease remains scarce thus
far.”
Dauchet et al., Nat. Rev. Cardiol. 2009
1) Consumption of fruits and vegetables is weakly associated with the
risk of CHD in cohort studies
2) Prevention trials have failed to show clear effects of fruit and
vegetable consumption on the occurrence of CHD
3) Consumption of fruits and vegetables is associated with decreases
in the risk for high blood pressure, but effects on other CHD risk
factors has not been clearly established
4) Prevention trials have failed to confirm the hypothesis that vitamins
and other individual nutrients in fruits and vegetables prevent CHD
The inverse association seen between the
consumption of plant food-rich diets and the risk
for CVD is due to multiple nutritional variables:
favourable sodium/potassium profile
low in saturated fat
high in essential nutrients (Mg, vitamin C,
Vitamin E, etc )
high in fibre
high in select “non-essential” phytochemicals
Biomarkers are needed for the
assessing an individuals
status for the phytochemical(s)
in question, as well as for
following the acute, and
chronic impact of these
phytochemicals on select
health parameters
Evaluation of Biomarkers 2010-
The Biomarker Evaluation Process Recommendations
Recommendation 1:
The biomarker evaluation process should consist of the
following three steps:
1a. Analytical validation: analyses of available evidence
on the analytical performance of an assay;
1b. Qualification: assessment of available evidence on
associations between the biomarker and disease states,
including data showing effects of interventions on
both the biomarker and clinical outcomes; and
1c. Utilization: contextual analysis based on the specific
use proposed and the applicability of available evidence
to this use. This includes a determination of whether the
analytical validation and qualification conducted provide
sufficient support for the use proposed.
Evaluation of Biomarkers 2010-
The Biomarker Evaluation Process Recommendations
Recommendation 2:
2a. For biomarkers with regulatory impact, the FDA
should convene expert panels to
evaluate biomarkers and biomarker tests.
2b. Initial evaluation of analytical validation and
qualification should be conducted
separately from a particular context of use.
2c. The expert panels should reevaluate analytical
validation, qualification, and
utilization on a continual and a case-by-case basis.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010-
The Biomarker Evaluation Process Recommendations
Recommendation 3:
The FDA should use the same degree of scientific
rigor for evaluation of biomarkers
across regulatory areas, whether they are
proposed for use in the arenas of drugs,
medical devices, biologics, or foods and dietary
supplements.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010-
The Biomarker Evaluation Process Recommendations
Recommendation 4:
The FDA should take into account a nutrient’s or food’s
source as well as any modifying effects of the food or
supplement that serves as the delivery vehicle and the
dietary patterns associated with consumption of the
nutrient or food when reviewing health related
label claims and the safety of food and supplements.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010-
Strengthening Evidence-Based Regulation Recommendations
Recommendation 5:
5-a. Congress should strengthen the FDA’s authority to
request and enforce post market surveillance across
drugs, devices, and biologics when approvals
are initially based on putative surrogate endpoint data.
5-b. Congress should grant the FDA authority to
request studies and sufficient authority to act on the
results of studies on consumer understanding of claims
on foods and supplements.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010-
Strengthening Evidence-Based Regulation Recommendations
Recommendation 6:
6-a. The U.S. Department of Health and Human Services
should facilitate a coordinated, department-wide effort to
encourage the collection and sharing of data about
biomarkers for all uses, including drugs, biologics,
devices, and foods.
6-b. The FDA in coordination with other federal agencies
should build needed data infrastructure and surveillance
systems to handle the information necessary to gain
sufficient understanding of the effects of biomarker use.
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010Case Studies
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
Evaluation of Biomarkers 2010Case Studies
Evaluation of Biomarkers and Surrogate Endpoints in Chronic
Disease; IOM, 2010
But, prior to defining appropriate biomarkers
for these non-essential phytochemicals; we
need a list of likely candidates.
An analysis of 16 year follow-up dietary data from 34,489
women enrolled in the Iowa’s Women Heath Study
Mink et al. Am J Clin Nutr 2007; 85: 895-909
DIVERSITY IS THE RULE
Class
Structure
Flavonoid
Food
Examples
Flavanols
Flavanol
Catechin
Cocoa, Wine, Tea, Dill
Weed, Chinese
Cabbage
Flavonols
Flavonol
Quercetin
Onion, Apple, Kale,
Arugula, Asparagus
Flavones
Flavone
Apigenin
Celery Hearts, Parsley,
Seed-herb, spice
Naringenin
Citrus fruits
Genistein
Soy products
Delphinidin
Radishes,
Red Cabbage, Purple
Carrots
Flavanones
Flavanone
Isoflavones
Anthocyanidins
Anthocyanidin
Rate Ratios of CVD Mortality of Flavonoid
Intake for CVD-Free Postmenopausal
Women
Class
Q1
Q2
Q3
Q4
Q5
P for Trend
0.6 - 133
1.00
133 - 202
0.85
202 - 282
0.78
282 - 425
0.74
425 - 3524
0.79
0.005
15 - 29
0.76
29 - 136
0.77
136 - 1050
0.85
0.885
144 - 212
0.81
212 - 343
0.81
Total Flavonoids
Intake (mg/day)
RR
Flavan-3-ols or monomers
Intake (mg/day)
RR
0-7
1.00
7 - 15
0.75
Proanthocyanidins
Intake (mg/day)
RR
0.0 - 90
1.00
90 - 144
0.92
343 - 3226
0.79
0.002
Mink et al. Am J Clin Nutr 2007; 85: 895-909
Lowest Range of Flavonoid Intake for Reduced
Risk in CVD Free Postmenopausal Women
Total
Mortality
CHD
CVD
Total Flavonoids
Intake (mg/day)
RR (95% CI)
133.2 - 201.8
0.85 (0.79, 0.92)
133.2 - 201.8
0.81 (0.69, 0.96)
133.2 - 201.8
0.85 (0.75, 0.97)
Proanthocyanidins
(includes monomers of: apples, chocolate, seeded grapes)
Intake (mg/day)
RR (95% CI)
89.5 - 143.9
0.89 (0.83, 0.96)
143.9 - 212.3
0.79 (0.67, 0.94)
143.9 - 212.3
0.81 (0.71, 0.92)
Flavan-3-ols (apples, red wine, green tea, black tea)
No significant associations
Mink et al. Am J Clin Nutr 2007; 85: 895-909
Consumption of Flavanol-containing Beverage Products
- Uptake of Flavanols into the Circulation-
6 hours
2 hours
1 hours
0 hours
Epicatechin
Response (nA)
300
200
Catechin
100
0
Flavanol Stereoisomers
Blood Plasma Flavanols/Metabolites after eating a
high-flavanol cocoa product
Circulating Flavanols/Metabolites [nM]
3000
epicatechin
catechin
4'MEC
EC-7Gluc
4'MEC-Gluc
Sum
2500
2000
1500
1000
500
250
0
-1
0
1
2
3
4
5
6
7
time [h]
Schroeter et al. PNAS 103:1024-29,2006
FMD – Nitric Oxide – Flavanol/Metabolites
Flavanol-mediated increases
in FMD are inhibited by L-NMMA
infusion and NOS inhibition
b
12
4
*#
6
*#
*#
*#
4
2
3
Increase RXNO (nM)
Flow-mediated dilation (%)
10
8
c
2
0
*#
3,000
*#
1
#
0
-1
* *
2,500
*#
*#
2,000
1,500
1,000
*#
*#
*#
500
*
-2
0 1 2 3 4 5 6
hFCD
lFCD
3,500
Sum of plasma flavanols (nM)
a
0
0 1 2 3 4 5 6
*
**
0 1 2 3 4 5 6
Time after cocoa drink (h)
Schroeter et al. PNAS 2006
Epicatechin - FMD
b
d
10
*#
*#
PAT index
3
*
*
*
2
1
Water
1 mg/kg Epicatechin
0
Flow-mediated dilation (%)
4
*#
8
6
4
2
Water
1 mg/kg Epicatechin
0
0
0
1
2
3
*#
4
1
2
3
4
Time after Ingestion (h)
Time after Ingestion (h)
Schroeter et al. PNAS 103:1024-29,2006
How Does FMD/PAT Compare to Blood
Pressure as An Accepted Marker?
•
Predictive value of noninvasively determined endothelial
dysfunction for long-term cardiovascular events and
restenosis in patients undergoing coronary stent
implantation: a prospective study.
Akcakoyun M, et. al. Coron Artery Dis, 2008; 19:337-343
•
Persistent impairment of endothelial vasomotor function
has a negative impact on outcome in patients with coronary
artery disease.
Kitta Y, et. al., J Am Coll Cardiol 2009;53:323-30
Assessment of Peripheral Vascular Endothelial
Function in the Ambulatory Setting
CAD+ n = 31
CAD- n = 29
Kuvin, JT et al. Vasc Med 12:1312:13-16, 2007
Rubinshtein, R et al. Eur Heart J 31:114231:1142-8, 2010
Cardiovascular Adverse Events in Patients with
or without Low L-RHI (natural log of PAT ratio)
Rubinshtein, R et al. Eur Heart J 31:114231:1142-8, 2010
PAT Gender Differences
Hamburg, NM et al. Circulation 117:2467117:2467-74, 2008
Flavanol Steroisomers
FA diameter (Δ%)
A
C 15
*
10
5
B
B
15
FA diameter (Δ%)
Stereospecific Effects of Flavanols on Arterial Dilation in a
Rodent Model (Momma et al., unpublished)
10
*
5
0
-5
0
(-)
(+)
Catechin
(+)
(-)
Epicatechin
Saline
+
(-)Epicatechin L-NMMA
-
+
-
+
+
+
Is there evidence of a similar specificity in humans?
Influence of stereochemistry on
plasma flavanol levels in humans
1400
Sum of flavanol metabolites ± SD (nM)
a
1200
1000
Control
(-)-Epicatechin
(+)-Epicatechin
(+)-Catechin
(-)-Catechin
d
b
800
b
e
600
e,c
400
c
200
c
n.d.
0
0
n.d.
2
Time post-consumption (h)
(Ottaviani et al unpublished)
4
Influence of stereochemistry on
flavanol metabolism in humans
Plasma flavanol metabolites
2 h post-consumption (nM)
1200
p<0.05
non-methylated metabolites
3'-O-methylated metabolites
4'-O-methylated metabolites
1000
800
600
400
200
n.d.
0
(-)-epicatechin
(+)-epicatechin
(-)-catechin
n.d.
(+)-catechin
• Catechin enantiomers did not yield 4′-O-methylated metabolites
• Epicatechin enantiomers differed in the levels of non-methylated metabolites
Cocoa Processing Effects on Flavanol
Epimerization
Non-alkalized
Alkalized
Ritter C et al. Anal Bioanal Chem 397:723397:723-30, 2010
Time Course of (-) Catechin Formation When
Tea is Heated
(-) Epicatechin degradation
(-) Catechin formation
50
70o C
80o C
100
40
Concentration %
Concentration %
90o C
50
30
20
70o C
80o C
10
10
90o C
0
0
20
40
60
Time, min
80
100
120
0
20
40
60
80
100
120
Time, min
Gotti et al, Electrophoresis, 2009
Effects of Flavanols and Flavanol-Rich Food
Consumption on Vascular Function
Reference
Type
Dose
Duration
Subjects
n
Vascular Reactivity
Engler ‘04
Chocolate
213 mg / dy
2 wks
Healthy adults
21
Increase FMD
Vlachopoulos
‘05
Chocolate
100g chocolate
1 dose
Healthy adults
17
Increase FMD, PWV
Heiss ‘05
Cocoa beverage
180 mg
1 dose
Smokers
11
Increase FMD
Grassi ‘05
Chocolate
88 mg
2 wks
Hypertensive &
Healthy Controls
20
Increase FMD both
groups
Schroeter ‘06
Epicatechin
1-2 mg/kg
1 dose
Healthy adults
3
Fisher ‘06
Cocoa Beverage
821 mg
4 days
Healthy adults
27
Increase PAT
Heiss ‘07
Cocoa Beverage
918
1 week
Smokers
6
Increase FMD
Flammer ‘08
Chocolate
36 mg
1 dose
Heart transplant
44
Increase Coronary
Reactivity
Balzer ‘08
Cocoa
Up to 960 mg
30 days
Diabetics
41
Increase FMD
Muniyappa ‘08
Cocoa
~ 900 mg
2 wks
Hypertensive
20
Shiina ‘09
Chocolate
550 mg
2 wks
Healthy Adults
39
Increase FMD, PAT
Increase Insulin
stimulated FMD
Increase Coronary
Flow Velocity
Can changes in FMD be associated with changes in
well accepted vascular health biomarkers?
(750 mg flavanols/procyanidins for 30 days)
Heiss et al. JACC In Press
Epidemiology evidence that high flavanol diets are
associated with a reduced risk for hypertension.
Hollenberg, NK J Cardiovasc Phamacol 2006
Urine can reflect epicatechin intake
a
Urinary epicatechin
equivalents (nΜ )
Urinary nitrate/nitrite
(μ mol/mmol creatinine)
70
700
60
600
50
500
40
400
* *
30
300
20
200
10
100
0
0
Ailigandi
Kuna Nega
Schroeter et al., PNAS 103: 1024-29, 2006
Urinary Flavanol Metabolite Concentrations 24 h
After Cocoa Consumption
Urpi-Sada et al., J Chromatogr A, 2009
Effects of Flavanols and Flavanol-Rich Food
Consumption on Blood Pressure
Reference
Type
Dose
Duration
Subjects
n
Blood Pressure
Engler 2004
Chocolate
213 mg / dy
2 wks
Healthy adults
21
No Change
Grassi 2005
Chocolate
~ 500 mg
polyphenols
2 wks
Healthy adults
15
Decreased Systolic
Grassi 2005
Cocoa beverage
88 mg
2 wks
Hypertensive
20
Decreased
Taubert 2007
Chocolate
18 weeks Pre ÆStage 1
hypertensive
44
Decreased
Davison 2008
Cocoa Beverage
902 mg
12 wks
49
Decreased Diastolic, MAP
Grassi 2008
Chocolate
150 mg
2 wks
Overweight/
Obese
Hypertensive
Balzer 2008
Cocoa
960 mg
30 dys
Medicated
Diabetics
41
No Change
Faridi 2008
Chocolate
821 mg
1 dose
Healthy
45
Decreased
Manuiyappa 08
Cocoa
~ 900 mg
2 wk
Hypertensive
20
No Change
Shiina 2009
Chocolate
550 mg
2 wks
Healthy Adults
39
No Change
30 mg /day
19
Decreased 24 hr
ambulatory BP
On an acute basis, dose dependent effects can be shown
Acute Effects from the Feasibility Trial ( 75, 371, 963 mg)
Balzer et al., J Am Coll Cardio, 51: 2141-9, 2008
What are the dose dependent effects over time?
Efficacy Trial: 321 mg flavanols per day - 3 times per day
versus 25 mg - 3 times per day
Thus, the total daily dose was 963 mg versus 75 mg
Balzer et al., J Am Coll Cardio, 51: 2141-9, 2008
(What are appropriate controls/placebos?)
Taubert D et al., JAMA 298: 49-60, 2007
Flavonoids & Vascular Health
Speculation:
The inverse association seen between the
consumption of plant food-rich diets and the risk
for CVD is due to numerous factors including
flavonoid-induced changes in: oxidant
production and defence; membrane
structure/function; the immune system; platelet
reactivity, blood pressure, tissue repair and
vascular reactivity.
Effects of Flavonoids and Flavonoid-Rich
Food Consumption on Platelet Function
Reference
Type
Dose
Duration
Subjects
n
Platelet aggregation
PacePace-Asciak ‘96
Red wine
White wine
Purple grape juice
375 ml/day
375 ml/day
500 ml/day
4 wks
Healthy adults
24
ª
-ª
Freedman ‘01
Purple grape juice
7 ml/kg/day
2 wks
Healthy adults
20
ª
Rein ‘00
Cocoa beverage
300 ml
1 dose
Healthy adults
10
ª
Platelet Reactivity
Hubbard ‘04
QuercetinQuercetin-4’-B-Oglucoside
150 mg
1 dose
Healthy adults
6
ª
Collagen
stimulated platelet
aggregation
Heptinstall ‘06
Cocoa Beverage
600600-900 mg
1 dose
Healthy adults
12
ª
Collagen
stimulated Platelet
Aggregation
Polagruto ‘07
Grapeseed Extract
400 mg
1 dose
Smokers
13
ª
Platelet Reactivity
Flammmer ‘07
Chocolate
40 g
1 dose
Heart
transplant
28
ª
Platelet adhesion
ª
Platelet Reactivity
ª
Platelet Reactivity
Hamed ‘08
Chocolate
100 g / day
1 week
Healthy adults
Erlund ‘08
“Berries”
Berries”
100 g
8 wks
Health Adults
72
Reported *Flavanol-Rich Cocoa/Chocolate
Intakes for Select Cardiovascular Effects
Time period
Dose (mg)*
Outcome Measure
Platelet Reactivity
1 day – 4 weeks
Blood Pressure
1 day – 18 weeks
30 - 900
Vascular Function
1 day – 4 weeks
88 - 960
* flavanol + procyanidins
** chocolate with an undefined flavanol content
**undefined – 900
Mechanisms?
The effect of 220 mg of flavonoids on the
ratio of prostacyclin (PGI2) to leukotriene (LT).
PGI2/LT Ratio
8.0
6.0
*
*
* P < 0.05
4.0
2.0
0.0
0h
2h
6h
Schramm et al, AJCN, 2001:73:36 & Holt et al, JAMA, 2002;287:2212
5-lipoxygenase inhibition ?
NADPH oxidase inhibition ?
Schewe et al, AJCN, 2005;81:304-12S
Schewe et al, Arch Biochem Biophys, 2008;15:102-6
Flavanol Intervention Mobilizes Functional
CACs
Circulating Endothelial Progenitor Cells,
Vascular Function, and Cardiovascular Risk
Hill, JM et al. NEJM 348:593348:593-600, 2003
Do high flavonoid diets influence the immune system?
Four Weeks of Cocoa Consumption (~ 500 mg
flavonoids/day) Reduces
Circulating Inflammatory Markers
Compared to Baseline and Milk only
sP-Selectin ↓ p 0.31
sICAM-1
↓ p 0.34
MonocyteVLA-4
↓ p 0.39
CD40
↓ p 0.31
Monagas et al, Am J Clin Nutr, 2009;90:1144-50
Contribution of 30 Biomarkers to 10-Year Cardiovascular Risk
Estimation in 2 Population Cohorts: The MONICA, Risk, Genetics,
Archiving, and Monograph (MORGAM) Biomarker Project
Blackenberg, S et al. Circulation 121:2388121:2388-97, 2010
Summary:
Collectively, the results obtained from numerous trials support
the concept that the consumption of select plant food products
can result in acute (1 – 30 days) improvements in vascular health
parameters that are suggestive of a decreased risk for select
chronic diseases.
The results obtained to date from numerous epidemiological
studies are consistent with the above concept.
Accumulating evidence supports the idea that in certain cases,
specific non-essential nutrients are driving the positive health
effects associated with some plant foods.
Established, and validated, biomarkers for an individuals status
of the above non-essential nutrients are largely lacking.
Summary:
When considering the development of biomarkers for
following the effects of a food or a non-essential
nutrient on the risk for select diseases, consideration
should be given to using a multiple component model
that takes into consideration the potential effects of
the food/nutrient on multiple factors.