Perspective

Received: 17 January 2012 Accepted: 17 January 2012 Published online in Wiley Online Library: 20 February 2012

(wileyonlinelibrary.com) DOI 10.1002/jsfa.5631

Nutritional biomarkers for objective dietary

assessment
Gunter GC Kuhnle∗

Abstract
The accurate assessment of dietary exposure is important in investigating associations between diet and disease. Research
in nutritional epidemiology, which has resulted in a large amount of information on associations between diet and chronic
diseases in the last decade, relies on accurate assessment methods to identify these associations. However, most dietary
assessment instruments rely to some extent on self-reporting, which is prone to systematic bias affected by factors such as age,
gender, social desirability and approval. Nutritional biomarkers are not affected by these and therefore provide an additional,
alternative method to estimate intake. However, there are also some limitations in their application: they are affected by
inter-individual variations in metabolism and other physiological factors, and they are often limited to estimating intake of
specific compounds and not entire foods. It is therefore important to validate nutritional biomarkers to determine specific
strengths and limitations. In this perspective paper, criteria for the validation of nutritional markers and future developments
are discussed.

c 2012 Society of Chemical Industry

Keywords: biomarker; nutrition; nutritional epidemiology; diet assessment

INTRODUCTION NUTRITIONAL BIOMARKERS

Lifestyle factors such as smoking, alcohol consumption, phys-
ical activity and diet are important modifiable risk factors for
chronic diseases, with an up to fourfold difference in mortal-
ity risk – equivalent to 14 years – between extremes of healthy
behaviour.1 Nutritional epidemiology has provided a large amount
of data on associations between diet and health, and these
findings are used to give advice to the public, for example
to reduce the risk of cancer.2 However, for many foods the
data available are inconclusive: for example, the beneficial ef-
fect of fruit and vegetables was considered to be convincing
in the comprehensive review of the World Cancer Research of
1997;3 later reports downgraded this assessment.4 – 6 A possible
explanation for this lack of strong association is that measure-
ment errors associated with dietary assessment attenuated the
results.7,8
Nutritional epidemiology relies on accurate dietary information
to investigate associations between diet and disease risk; most
dietary assessment instruments rely to some extent on self-
reporting, which is prone to systematic bias affected by factors such
as age, gender, social desirability and approval.9,10 Self-reporting
by FFQ (food–frequency questionnaire), for example, is known to
result in over-reporting of fruit and vegetable intake,11 whereas
obese people are known to under-report total energy,12 protein12
and sugar intake.13 Fig. 1 illustrates the differences between self-
reported and biomarker data of sugar intake. Statistical models
developed to accommodate these errors require at least two
exposure assessments with unrelated measurement error, and
this cannot be achieved using methods based on self-reporting
alone. Nutritional biomarkers, however, can provide such a
The term biomarker has several different meanings; most com-
monly, biomarkers are used as surrogate endpoints in clinical
studies and are therefore used to predict future events; con-
versely, biomarkers of exposure are used to determine past events.
Nutritional biomarkers, which are used to establish past dietary
intake, belong to the latter group and are the sole focus of this
article.
Nutritional biomarkers are indicators of dietary exposure;
therefore any parameter which is associated with exposure
and can be measured objectively can be used as a nutritional
marker. Most commonly, compounds found in foods – and their
metabolites – are used as biomarkers, although physical properties
such as stable isotope ratio are also suitable. Depending on the
relationship between intake and biomarker, they are divided
into three main classes: recovery biomarkers are based on the
total excretion of the marker over a defined time period. They
require that excretion is a fixed proportion of intake in any
individual with only negligible inter-individual variation, and their
development is based on the knowledge of physiological balance
between intake and excretion.15,16 For this reason they are best
suited to estimate absolute intake, but only few recovery markers
exist (urinary nitrogen and potassium). Concentration markers are
based solely on the concentration of the respective marker; no
information on the physiological balance of intake and excretion
∗
Correspondence to: Gunter GC Kuhnle, Department of Food and Nutritional
Sciences, University of Reading, Reading RG6 6 AH, UK.
E-mail: g.g.kuhnle@reading.ac.uk
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Department of Food and Nutritional Sciences, University of Reading, Reading

method.14 RG6 6 AH, UK

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flavonoids21 or total polyphenols,22 as the lack of detailed food

Figure 1. Association of sugar intake and obesity using dietary and
biomarker data. Data from Bingham et al.32
is necessary, and this balance can differ considerably between
individuals. For this reason, they cannot be used to estimate
absolute intake and additional information is required to provide
a reference.16 However, since they are correlated with intake, they
can be used to rank intake of specific nutrients. A further class of
biomarkers are predictive biomarkers, which have been proposed
by Tasevska et al. for biomarkers with incomplete recovery but a
stable and time-dependent high correlation with intake.17 In their
ability to estimate absolute intake, these markers rank between
concentration and recovery markers. The only predictive biomarker
available at the moment is urinary sucrose and fructose as marker
of sugar intake.18
LIMITATIONS OF BIOMARKERS
Nutritional biomarkers allow the objective assessment of dietary
exposure, either directly using recovery markers, or indirectly in
combination with self-reported intake,19 and there is abundant ev-
idence for the importance of nutritional biomarkers in nutritional
epidemiology. Biomarkers are, in particular, useful in estimating in-
take of specific nutrients or compounds such as phytoestrogens,20
composition data and the large variability within foods23 make it
difficult to estimate exposure otherwise. However, this is also one
of the key limitations of nutritional biomarkers: it is very difficult to
identify the source of these compounds using biomarkers alone,
except for compounds which are specific to only a certain type
of food. The physiological effect of food is not limited to indi-
vidual compounds, but is based on their combination as well as
other factors such as macronutrient content and energy density.
These aspects are often neglected when focusing on individual
compounds as biomarkers.
A further limitation of biomarkers is the difficulty in assessing
intake of food groups. Fruits and vegetables are believed to
have a beneficial effect, but assessment of intake remains difficult.
Despite extensive research in the past decade for good biomarkers
of intake, vitamin C and carotenoids remain the most commonly
used ones.24 This voluntary restriction to a small number of specific
compounds introduces a considerable bias when assessing diet:
when using vitamin C as biomarker of fruit and vegetable intake, a
portion (80 g) of green pepper is equivalent to 20 portions (1.6 kg)
of carrots – however, when using carotenoids as biomarker, one
portion of carrots is equivalent to more than 45 portions of
green peppers (see Table 1 for details). Unless vitamin C or
carotenoids are the main bioactive ingredient, these differences
will affect conclusions and lead to skewed results. To address this
problem, it is important either to use a combination of biomarkers,
as recommended previously,24 or to develop new biomarkers
based on different compounds, for example total phenols, which
have a much lower variation across different types of fruits and
vegetables.22
Intra- and inter-individual differences in absorption and
metabolism also affect the actual or measured concentration
of biomarkers in plasma and urine, and thereby the biomarker-
derived assessment of exposure. Absorption of specific biomarkers
depends not only on the composition of the diet but also on ge-
netic variability.25 Differences in metabolism not only have an
effect on circulating levels of biomarkers but also affect biomarker
analysis. Many compounds used as biomarkers, such as polyphe-
nols, undergo extensive metabolism, both by the gastrointestinal
microbiome26 and the liver and elsewhere;27 although many
of these metabolites are still structurally related to the original

Table 1. Vitamin C, carotenoids and total phenol content of different fruits and vegetables commonly consumed in the UK, and number of portions
(80 g) required to achieve mean intake of the respective biomarker. Data from McCance and Widdowson35 and the USDA database36

Vitamin C Carotenoids Total phenols

(mg 100 g−1 ) Portions (µg 100 g−1 ) Portions (mg 100 g−1 ) Portions

Apple 6 2.3 18 2.3 202 1.1

Banana 11 1.3 21 2.0 155 1.4
Beetroot 5 2.8 27 1.6 164 1.3
Carrots 6 2.3 12 472 – 58 3.7
Cucumber 2 6.9 60 0.7 21 10.2
Kiwi 59 0.2 40 1.0 180 1.2
Onion 10 1.4 5 8.4 103 2.1
Orange 54 0.3 47 0.9 279 0.8
Pepper (green) 120 0.1 265 0.2 229 0.9
Pepper (red) 140 0.1 3 840 – 182 1.2
Potato 11 1.3 0 ∞ 54 4.0
Strawberry 77 0.2 8 5.2 289 0.7
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compound, analytical methods used for biomarker analysis often

fail to detect most of them,28 thus introducing an additional bias,
in particular since differences in metabolism can also be associated
with disease risk.
A further limitation of many existing biomarkers is their short
physiological half-life, in particular when measured in specimens
such as plasma or urine. Many phenolic compounds are excreted
within less than 24 h, thus providing information about this period
only. Even for lipophilic compounds such as carotenoids the half-
life is less than 100 days;29 this is shorter than the time covered by
some FFQs.

BIOMARKER DEVELOPMENT: DISCOVERY

AND VALIDATION
The development of new biomarkers for dietary assessment is
crucial not only for nutritional research but also for clinical
practice, for example to monitor compliance in patients. The
development of new biomarkers consists of two parts: discovery
and validation. Several strategies for biomarker discovery have
been proposed, but they can be broadly divided into two different
approaches, as summarized in Fig. 2: in a so-called hypothesis
driven approach, information about compounds believed to be
linked to the intake of specific foods, such as food components or
specific contaminants, are investigated as candidate biomarkers.
This approach requires detailed knowledge of food constituents
and the effects of preparation, as well as bioavailability and
metabolism; the most common metabolites are often chosen as
candidate biomarkers, and analytical methods are developed for
their analysis. Most of the biomarkers currently available have
been developed using this method. The second, discovery-driven,
approach has become more important with the advent of so-
called metabolomics techniques. In this approach, candidate
biomarkers are identified in samples from carefully controlled
human intervention studies using multivariate techniques. Proline
Figure 2. Hypothesis-driven and discovery-driven development strategies
betaine – a biomarker of citrus fruit consumption – is one of for nutritional biomarkers.
the first biomarkers developed using this technique.30 The
advantage of the discovery-driven approach is that much less
a priori knowledge is required as the candidate biomarker is 2. The suitability of the candidate biomarker in a free-living
identified using statistical methods. However, it requires very population should be investigated using a (controlled) habitual
carefully controlled dietary intervention and sample collection diet.
protocols,31 as the multivariate techniques used to identify
These two validation steps often raise further questions which
candidate biomarkers are very sensitive to any differences found
will require additional studies, for example to investigate whether
in the sample.
the biomarker is applicable to a specific group of the population.
An important, and often neglected, aspect of biomarker
Urinary sucrose and fructose as biomarker of sugar intake have
development is biomarker validation. In contrast to predictive
been validated using a study to investigate the dose–response
biomarkers, for which validation is complicated as they attempt to
effect and a study with a controlled habitual diet.18 As the
predict future events, the validation of biomarkers of exposure is
application of these biomarkers showed a considerable difference
less difficult, in particular when exposure can be easily controlled.
between obese and normal weight subjects,32 further validation
Dietary exposure can be easily controlled in dietary intervention
studies were conducted to investigate these differences33 and
studies, where subjects consume a controlled diet. Ideally, a
confirm that these biomarkers are not restricted to a normal-
nutritional biomarker should be validated in two stages:
weight population.
1. The dose–response effect should be investigated in dietary The validation of a candidate biomarker in controlled dietary
intervention studies where participants receive identical foods intervention studies does not only provide information about
except for the food under investigation. This will provide correlation with intake, but also information about potential
information on the association between food and candidate limitations such as range of intake and interactions with other
biomarker that is not affected by background diet, and it allows foods. It is therefore important for the development of new
identification of the range of intake for which a candidate biomarkers; however, most biomarkers are currently validated
biomarker is suitable, as some compounds – for example, using self-reported intake data only, despite the known limitations
vitamin C18 – show saturation and might therefore not be of these assessment methods. This makes it very difficult to
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suitable to determine high intake. assess the reliability of a candidate biomarker, as a non-significant

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Figure 3. Time frame (not to scale) covered by different dietary assessment
methods.
correlation with intake could be explained either by a poorly
performing biomarker or by inaccurate dietary assessment.
CONCLUSIONS AND OUTLOOK
Nutritional biomarkers are important for future research into
associations between diet and health, as they provide an objective
assessment method for dietary exposure. There is currently a
considerable demand for the development of new markers, for
example for the objective assessment of fruit and vegetable intake.
The introduction of new technologies, in particular metabolomics,
has provided new and useful tools for the development of new
candidate biomarkers, and the results so far are very promising.
However, it is important that new candidate biomarkers are
validated using carefully controlled dietary intervention studies,
and not just self-reported dietary data. There is currently no formal
guideline for the validation of nutritional biomarkers, but the
following criteria could be used as an indicator:
• There should be an explanation for the correlation between
intake and biomarker; e.g. the biomarker should be a
metabolite of a food component.
• A clear dose–response relationship should be established
using dietary intervention studies. This should be confirmed in
a study with a controlled habitual diet to ensure a wide variety
of different foods.
• There should be detailed information about the limitations
of the biomarker, such as saturation effects or limitation to a
subgroup of the population.
• There should be information about the analytical methods
used to analyse the biomarker.
The development of a repository for nutritional biomarkers,
including details on their correlation with intake, analytical
methods and limitations, would also facilitate their development
and in particular their validation. Although several systematic
reviews on nutritional biomarkers exist,24,34 so far there is no
comprehensive database available.
A further aspect of nutritional biomarkers that should be
addressed is the limited time window of assessment. Biomarkers
currently used are mainly restricted to the assessment of acute
intake, in particular because of the specimen collected and used for
analysis. Alternative specimen such as hair can provide an option
to obtain information on long-term intake or chronic exposure
(Fig. 3), and although they are used to some extend in nutritional
research they are not commonly used in nutritional epidemiology.
New nutritional biomarkers will facilitate future research
into associations between diet and disease and provide new
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information about the health effects of food. This will not only
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GGC Kuhnle
allow a better substantiation of health claims but also provide the
general public with better dietary advice.
REFERENCES
1 Khaw K-T, Wareham N, Bingham S, Welch A, Luben R and Day N,
Combined impact of health behaviours and mortality in men and
women: the EPIC–Norfolk Prospective Population Study. PLoS Med
5:e12 (2008).
2 World Cancer Research Fund/American Institute for Cancer Research,
Food, Nutrition, Physical Activity, and the Prevention of Cancer: A
Global Perspective. AICR, Washington, DC (2007).
3 World Cancer Research Fund/American Institute for Cancer Research,
Food, Nutrition and the Prevention of Cancer: A Global Perspective.
World Cancer Research Fund, London (1997).
4 IARC Working Group on the Evaluation of Cancer-Preventive
Strategies, Fruit and Vegetables. IARC Press, Lyon (2003).
5 World Cancer Research Fund International, Food, Nutrition, Physical
Activity, and the Prevention of Cancer: A Global Perspective. A project
of World Cancer Research Fund International. American Institute
for Cancer Research, Washington, DC (2007).
6 Boffetta P, Couto E, Wichmann J, Ferrari P, Trichopoulos D, Bueno-de-
Mesquita HB, et al, Fruit and vegetable intake and overall cancer risk
in the European Prospective Investigation Into Cancer and Nutrition
(EPIC). J Natl Cancer Inst 102:529–537 (2010).
7 Schatzkin A and Kipnis V, Could exposure assessment problems give
us wrong answers to nutrition and cancer questions? J Natl Cancer
Inst 96:1564–1565 (2004).
8 Jenab M, Slimani N, Bictash M, Ferrari P and Bingham SA, Biomarkers
in nutritional epidemiology: applications, needs and new horizons.
Hum Genet 125:507–525 (2009).
9 Hebert JR, Clemow L, Pbert L, Ockene IS and Ockene JK, Social
Desirability bias in dietary self-report may compromise the validity
of dietary intake measures. Int J Epidemiol 24:389–398 (1995).
10 Hebert JR, Ma Y, Clemow L, Ockene IS, Saperia G, Stanek EJ III, et al,
Gender differences in social desirability and social approval bias in
dietary self-report. Am J Epidemiol 146:1046–1055 (1997).
11 Bingham SA, Dietary assessments in the European prospective study
of diet and cancer (EPIC). Eur J Cancer Prev 6:118–124 (1997).
12 Lissner L, Troiano RP, Midthune D, Heitmann BL, Kipnis V, Subar AF,
et al, OPEN about obesity: recovery biomarkers, dietary reporting
errors and BMI. Int J Obes (Lond) 31:956–961 (2007).
13 Bingham S, Luben R, Welch A, Tasevska N, Wareham N and Khaw KT,
Epidemiologic assessment of sugars consumption using bio-
markers: comparisons of obese and nonobese individuals in
the European prospective investigation of cancer Norfolk. Cancer
Epidemiol Biomarkers Prev 16:1651–1654 (2007).
14 Prentice RL, Sugar E, Wang CY, Neuhouser M and Patterson R, Research
strategies and the use of nutrient biomarkers in studies of diet and
chronic disease. Public Health Nutr 5:977–984 (2002).
15 Freedman LS, Midthune D, Carroll RJ, Tasevska Na, Schatzkin A,
Mares J, et al, Using regression calibration equations that combine
self-reported intake and biomarker measures to obtain unbiased
estimates and more powerful tests of dietary associations. Am
J Epidemiol 174:1238–1245 (2011).
16 Kaaks R, Ferrari P, Ciampi A, Plummer M and Riboli E, Uses and
limitations of statistical accounting for random error correlations, in
the validation of dietary questionnaire assessments. Public Health
Nutr 5:969–976 (2002).
17 Tasevska Na, Midthune D, Potischman N, Subar AF, Cross AJ,
Bingham SA, et al, Use of the predictive sugars biomarker to
evaluate self-reported total sugars intake in the Observing Protein
and Energy Nutrition (OPEN) study. Cancer Epidemiol Biomarkers
Prev 20:490–500 (2011).
18 Tasevska N, Runswick SA, McTaggart A and Bingham SA, Urinary
sucrose and fructose as biomarkers for sugar consumption. Cancer
Epidemiol Biomarkers Prev 14:1287–1294 (2005).
19 Ocke M and Kaaks R, Biochemical markers as additional measurements
in dietary validity studies: application of the method of triads with
examples from the European Prospective Investigation into Cancer
and Nutrition. Am J Clin Nutr 65:1240S–1245S (1997).
20 Ward H, Kuhnle GGC, Mulligan AA, Lentjes MAH, Luben RN, Khaw K-T,
et al, Breast, colorectal and prostate cancer risk in EPIC-Norfolk in
relation to phytoestrogen intake using an improved database. Am
J Clin Nutr 91:440–448 (2009).
J Sci Food Agric 2012; 92: 1145–1149
Nutritional biomarkers for objective dietary assessment
21 Krogholm KS, Bredsdorff L, Alinia S, Christensen T, Rasmussen SE and
Dragsted LO, Free fruit at workplace intervention increases total
fruit intake: a validation study using 24 h dietary recall and urinary
flavonoid excretion. Eur J Clin Nutr 64:1222–1228 (2010).
22 Medina-Remon A, Barrionuevo-Gonzalez A, Zamora-Ros R, Andres-
Lacueva C, Estruch R, Martinez-Gonzalez MA, et al, Rapid
Folin–Ciocalteu method using microtiter 96-well plate cartridges
for solid phase extraction to assess urinary total phenolic
compounds, as a biomarker of total polyphenols intake. Anal Chim
Acta 634:54–60 (2009).
23 Kuhnle GGC, Dell’Aquila C, Runswick SA and Bingham SA, Variability of
phytoestrogen content in foods from different sources. Food Chem
113:1184–1187 (2009).
24 Baldrick FR, Woodside JV, Elborn JS, Young IS and McKinley MC,
Biomarkers of fruit and vegetable intake in human intervention
studies: a systematic review. Crit Rev Food Sci Nutr 51:795–815
(2011).
25 Jenab M, Slimani N, Bictash M, Ferrari P and Bingham SA, Biomarkers
in nutritional epidemiology: applications, needs and new horizons.
Hum Genet 125:507–525 (2009).
26 Rechner AR, Smith M, Kuhnle GGC, Gibson G, Debnam E, Srai S et al,
Colonic metabolism of dietary polyphenols: influence of structure
on microbial fermentation products. FreeRadicBiolMed 36:212–225
(2004).
27 Ottaviani JI, Momma TY, Kuhnle GK, Keen CL and Schroeter H,
Structurally related (−)-epicatechin metabolites in humans:
assessment using de novo chemically synthesized authentic
standards. Free Radic Biol Med (in press).
28 Ward HA, Kuhnle GGC, Mulligan AA, Lentjes MAH, Luben RN and
Khaw K-T, Breast, colorectal, and prostate cancer risk in the
European Prospective Investigation into Cancer and Nutrition–
Norfolk in relation to phytoestrogen intake derived from an
improved database. Am J Clin Nutr 91:440–448 (2010).
J Sci Food Agric 2012; 92: 1145–1149

c 2012 Society of Chemical Industry
www.soci.org
29 Burri BJ, Neidlinger TR and Clifford AJ, Serum carotenoid depletion
follows first-order kinetics in healthy adult women fed naturally low
carotenoid diets. J Nutr 131:2096–2100 (2001).
30 Heinzmann SS, Brown IJ, Chan Q, Bictash M, Dumas M-E, Kochhar S,
et al, Metabolic profiling strategy for discovery of nutritional
biomarkers: proline betaine as a marker of citrus consumption.
Am J Clin Nutr 92:436–443 (2010).
31 Lloyd AJ, Favé G, Beckmann M, Lin W, Tailliart K, Xie L, et al, Use of
mass spectrometry fingerprinting to identify urinary metabolites
after consumption of specific foods. Am J Clin Nutr 94(4):981–91
(2011).
32 Bingham SA, Luben RN, Welch AA, Tasevska N, Wareham NJ and
Khaw K-T, Epidemiologic assessment of sugars consumption using
biomarkers: comparisons of obese and nonobese individuals in the
European prospective investigation of cancer Norfolk. Am J Clin Nutr
16:1651–1654 (2007).
33 Joosen AMCP, Kuhnle GGC, Runswick SA and Bingham SA, Urinary
sucrose and fructose as biomarkers of sugar consumption:
comparison of normal weight and obese volunteers. Int J Obes
32:1736–1740 (2008).
34 Pérez-Jiménez J, Hubert J, Hooper L, Cassidy A, Manach C,
Williamson G, et al, Urinary metabolites as biomarkers of polyphenol
intake in humans: a systematic review. Am J Clin Nutr 92:801–809
(2010).
35 Holland B, Welch A, Unwin ID, Buss DH, Paul AA and Southgate DAT,
McCance and Widdowson’s The Composition of Foods. Royal Society
of Chemistry and Ministry of Agriculture, Fisheries and Food, London
(1991).
36 US Department of Agriculture, USDADatabasefortheFlavonoidContent
of Selected Foods. USDA, Washington, DC (2003).
1149
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