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Biomarkers of carotenoid bioavailability
Accepted Manuscript Biomarkers of carotenoid bioavailability
F. Granado-Lorencio, I. Blanco-Navarro, B. Pérez-Sacristán PII: DOI: Reference:
S0963-9969(17)30138-2 doi: 10.1016/j.foodres.2017.03.036 FRIN 6643
To appear in:
Food Research International
Received date: Revised date: Accepted date:
19 November 2016 15 March 2017 19 March 2017
Please cite this article as: F. Granado-Lorencio, I. Blanco-Navarro, B. Pérez-Sacristán , Biomarkers of carotenoid bioavailability. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/j.foodres.2017.03.036
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ACCEPTED MANUSCRIPT Biomarkers of carotenoid bioavailability Granado-Lorencio, F1 2,3*; Blanco-Navarro, I1,2,3; Pérez-Sacristán, B 1
Grupo Metabolismo y Nutrición, IDIPHIM;
2
1,2
Unidad de Vitaminas; 3 Servicio de Bioquímica Clínica,
Hospital Universitario Puerta de Hierro-Majadahonda, 28222 Madrid, Spain.
* Corresponding author: F. Granado-Lorencio
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Unidad de Vitaminas. Servicio de Bioquímica Clínica.
Hospital Universitario Puerta de Hierro-Majadahonda. 28222-Madrid (Spain) Telf: +34-911917578 / 7756
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Fax: +34-911916806
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e-mail: [email protected]
Abstract
The use of biomarkers constitutes an essential tool to assess the bioavailability of carotenoids in
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humans. The present article aims to review several methodological, host-related and modulating factors relevant on assessing and interpreting carotenoid bioavailability. Markers for carotenoid bioavailability can be broadly divided into direct, biochemical or “analytical” markers and indirect, physiological or “functional” indicators. Analytical markers usually refer to biochemical indicators of intake and/or status
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(short and long term exposure) while functional measures may be interpreted in terms of cumulative exposure, biological effect (bioactivity) or modification of risk factors. Both types of markers display
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advantages and limitations but, in general, a relationship exists among the type of marker, the biological specimen needed and the time required for a change. Humans may absorb a wide range of carotenes and xanthophylls and many of them may be found in serum and tissues. However, under physiological conditions, the several classes of dietary carotenoids may behave unequally leading to a different
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systemic profile and, moreover, they can be selectively accumulated at target tissues. In addition, some carotenoids may be chemically and enzymatically modified generating different oxidative metabolites and apocarotenoids. Quantitatively, the biological response upon carotenoid intervention (assessed by
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analytical and functional markers) is highly variable but the use of large doses and long-term protocols may lead to saturation effects and the loss of linearity in the response. Also, despite carotenoid exposition is considered to be safe, markers of overexposure include clinical signs (i.e. carotenodermia, corneal rings and retinopathy) and biochemical indicators (hypercarotenemia, xanthophyll esters). Overall, both host-related and methodological factors may influence analytical and functional markers to assess carotenoid bioavailability although the different subclasses of carotenoids may not be equally affected.
Keywords; Bioavailability, biomarkers, carotenoids, human study
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ACCEPTED MANUSCRIPT Index 1.- Introduction. 2.- Biomarkers 2.1.- Biomarkers of exposure 2.2. Functional biomarkers 3.- Methodological issues 3.1. Analytical points 3.2. Timing of intervention
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3.2.1. Post-prandial response, clearance and excretion 3.3. Biological matrix 4.- Markers of carotenoid bioavailability
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4.1.- Qualitative features
4.1.1.- In vivo metabolites as surrogate of carotenoid bioavailability
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4.2.- Quantitative considerations 4.3.- Markers of overexposure
5.- Modulating factors of markers of exposure and response
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6.- Concluding remarks
1.
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7.- References
INTRODUCTION
Carotenoids are fat-soluble pigments of plant origin that humans cannot synthesize although they can partially modify them. From a nutritional and physiological viewpoint, interest in carotenoids
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has been focused on the provitamin A activity of some of them but carotenoids also display other
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biological functions that may confer beneficial effects against chronic diseases. Prior to exerting their bioactivity, however, these compounds must be bioavailable. Bioavailability can be defined as the proportion of a dietary nutrient (or its metabolites) that is ultimately available for utilization or storage by target tissues after digestion, absorption and distribution (Ball, 1998).
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Nevertheless, because of the practical and ethical difficulties when measuring bioactivity and because of nutritional status also determine the amount of a nutrient that the body may use, store or excrete, the term “bioavailability” is usually referred to as the fraction of a given compound or its metabolites that
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reaches the systemic circulation without considering bioactivity (Holst & Williamson, 2008; CarbonellCapella et al, 2014).
In vitro models based on human physiology have been developed as simple and reproducible tools to study digestive processes (i.e. stability, micellization, intestinal transport) and to predict the bioavailability of different food components (i.e. carotenoids). Overall, reasonable correlations between in vitro bioaccessibility, in vivo observations and results from human bioavailability trials have been reported. Broadly, for lutein, β-carotene, lycopene and β-cryptoxanthin both qualitative and (semi)quantitative correlations have been found suggesting that in vitro bioaccessibility can be indicative of the amount available for uptake in the (in vivo) gastrointestinal tract (Reboul et al, 2006; GranadoLorencio et al, 2007a, 2007b; Maiani et al, 2009; Bohn et al, 2015). Caco-2 cells have been also used as surrogate for enterocytes and human absorption, including chylomicron secretion (Failla & Chitchumroonchokchai, 2005). Nevertheless, different Caco-2 cell lines may generate distinct metabolite
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ACCEPTED MANUSCRIPT profiles and these cells take up a wide range of carotenoids, even those not detected in human serum (i.e. neoxanthin, xanthophyll esters) (Sugawara et al. 2001; During et al, 2002; Failla & Chitchumroonchokchai, 2005), a fact that compromise its comparability with in vivo (human) situations. In general, the behaviour of carotenoids under in vitro gastrointestinal conditions does not fully explain the changes observed in vivo (Bohn et al, 2015). In vitro models may provide relevant information regarding food factors influencing bioavailability but they cannot totally simulate in vivo human situations, especially those concerning biological variability, timing of intervention, postprandial metabolism and distribution, and biological effects upon regular intake (Granado-Lorencio et al, 2011). Overall, carotenoid bioavailability in animals and humans have been approached using different methods
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including balance studies, dose-effect relationships and isotope-labeled compounds plus compartment modeling (Ball, 1998; Failla & Chitchumroonchokchai, 2005; Bohn, 2008). However, regardless of the method used, absorption, status, distribution and storage in humans are assessed with the use of
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biochemical markers.
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Within this context, the present article aims to review several methodological, host-related and modulating factors relevant on assessing and interpreting carotenoid bioavailability. We briefly describe approaches and variables affecting the assessment of the bioavailability of carotenoids in humans,
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including the type of markers, analytical issues, the time of exposure, qualitative and quantitative issues of the response as well as modulating and confounding factors affecting the interpretation of data. Overall, the paper provides an overview from a nutritional biochemistry perspective regarding the use of
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markers to assess carotenoid bioavailability. 2.- BIOMARKERS
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A key issue in the design of human nutritional studies is the choice of the right markers which, in turn, depends on the objective of the study. Overall, for essential nutrients, nutritional status (as a
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surrogate of bioavailability) can be approached by clinical, dietetic, anthropometric and biochemical markers. Nevertheless, for carotenoids, and especially for non-provitamin A carotenoids, most of these approaches are not applicable since nutritional status cannot be properly defined and inadequate intake
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does not result in biochemical or clinical signs of deficiency. As for other micronutrients and phytochemicals, biomarkers for carotenoid bioavailability can be broadly divided into direct, biochemical or “analytical” markers and indirect, physiological or “functional”
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indicators. Usually, analytical markers refer to biochemical indicators of intake and/or status (exposure) of a given nutrient or food component. In turn, functional markers evaluate the effect or a biological activity associated with a nutrient or its absence. 2.1.- Biomarkers of exposure Traditionally, the assessment of carotenoid exposure in humans has been performed by dietary or biochemical methods, both of which have advantages and limitations. In general terms, markers of exposure evaluate biological accessibility although a critical issue is to assess to what extent the marker reflects actual dietary exposure [Ellwood et al, 2014]. Overall, concentrations of carotenoids in serum and tissues are considered more reliable than dietary methods since they provide a more accurate measurement of the amounts available to tissues (Van den Berg et al, 2000; Maiani et al, 2009; Raiten et al, 2011; Scalbert et al, 2014; Tanumihardjo et al, 2016). However, serum carotenoids may be influenced by the bioavailability and in vivo metabolic changes and thus, they do not represent a true
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ACCEPTED MANUSCRIPT measurement of dietary exposure. Even so, blood concentrations of biomarkers provide direct relationships between nutrient input, status and availability for tissue metabolic requirements (Thurnham & Northrop-Clewes, 2016). Tissues also reflect exposure to carotenoids. Adipose tissue is thought to be a more stable depot of carotenoids although this requires invasive approaches (biopsies). Similarly, carotenoids accumulate in human skin and supplementation leads to increases in carotenoids content. Thus, skin carotenoid status (measured by reflectance methods or Raman spectroscopy) may be used as an objective biomarker of dietary intake (bioavailability), although diet explains only some of the variation in this biomarker (Mainke et al, 2010; Mayne et al, 2013). Also, non-invasive tests (i.e. heteroflicker
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photometry) have been used to measure macular pigment optical density (MPOD) which provides information on long-term lutein (and zeaxanthin) exposure (Beatty et al. 2001; Bernstein et al, 2002). other (non-ocular) tissues is uncertain (Granado et al, 2003).
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2.2.- Functional biomarkers
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However, because of the selective deposition of these carotenoids in this tissue (retina), its relevance to
Functional markers are considered to be indicative of biochemical/physiological changes as a
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result of the exposure. Overall, functional markers may be related with long-term (i.e. cumulative) exposure but they are subject to confounding factors and may be influenced by other micronutrients (i.e. methyl group donors and B vitamins for DNA methylation, interactions of divalent cations during
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absorption, copper/zinc-dependent enzyme activity) (Elmafda & Meyer, 2014). Noteworthy, many bioavailability studies with carotenoids are performed using “natural” sources (i.e. extracts, whole and fortified foods) rather than single compounds (i.e. supplements) so that other dietary components are co-ingested (collinearity) making difficult the interpretation of results in terms of comparability and
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“selectivity” (as a criteria of causality).
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Examples of functional tests used to evaluate carotenoid bioavailabity /response are shown in table 1. Under normal circumstances, the functional measures correspond to bioavailability, but the relationship may be influenced by host factors (Degeraud et al, 2015). Functional bioefficacy can be also measured by determining the rate by which deficiency symptoms are cured or by using other measures
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specific to the nutrient's function. As some carotenoid are converted into retinol, functional improvement in vitamin A status could be used as a surrogate of the intervention with carotenoids by assessing visual function or ocular indicators (Failla & Chinchorookchoktai, 2005; Degeraud et al, 2015). In addition,
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bioefficacy can be assessed using blood retinol concentration although serum retinol is homeostatically controlled by the liver (Tanumihardjo et al, 2016). In general, functional tests show several constraints such as the lack of standardization, the potential synergism between nutrients, the absence of accepted reference ranges or the influence of polymorphisms unrelated to the bioavailability of the nutrient (i.e. enzymes). Additionally, although these methods may provide an index of inadequacy (i.e. dark adaptation), they may not be responsive within the full range of normal intake or above [Raiten et al, 2011; Tanumihardjo, 2004]. Importantly, changes in markers of exposure or status (i.e. increase in serum levels) may not be necessarily followed by changes in markers of function or effect (i.e. reduction of markers of oxidative stress) and thus, both types of markers are not comparable or interchangeable since they provide different information regarding the bioavailability of a given compound (i.e. availability or associated biological action).
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ACCEPTED MANUSCRIPT 3.- METHODOLOGICAL ISSUES 3.1.
Analytical points
Naturally occurring dietary carotenoids include a wide range of chemical forms such as carotenes, free xanthophylls as well as mono- and di-esters of several xanthophylls mainly found in fruits (i.e. citrus fruits, papaya, goji berries) (Van den Berg et al, 2000; Maiani et al, 2009; Bohn et al, 2015). Although it is essential to identify the carotenoid profile in foods as it is what we may find in vivo (i.e. plasma, tissues), carotenoids may be metabolized in the body. In addition, during food processing, loss of carotenoids may occur and levels of cis-isomers may increase due to the isomerization of the trans-forms. However, many bioavailability studies are based on the measurement of intact (i.e. total
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and free forms) carotenoids in the blood or tissues and hence, so that characterization and quantification of carotenoid changes (i.e. cis-isomers, in vivo metabolites) is necessary to assess accurately
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bioavailability (Arathi et al, 2015) and the analytical approach becomes essential.
Overall, although the best approach may depend on the matrix and the objective of the study,
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analyses of carotenoids from biological matrices involves several steps including sample collection/ preparation, extraction (with or without saponification), separation, detection and quantification. However, despite new developments and improvements in separation techniques (i.e. columns, mass
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spectrometry detectors), analytical inaccuracies and artifacts (i.e. sample stability, extraction procedures, lack of standards) may be still present (van den Berg et al, 2000; Arathi et al, 2015; Bohn et al, 2015). In general, for food analyses and in vitro digestion systems, extraction with and without
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saponification may be used so that information regarding free forms and total content of xanthophylls may be obtained (i.e. to assess the degree of hydrolysis in vitro). A saponification step, however, is not usually performed with serum or triacylglycerol-rich lipoprotein fractions (TRL) due to the (expected)
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absence or very low presence of xanthophyll esters and the potential losses during the procedure. In clinical practice, carotenoids analysis (mostly β-carotene) is not routinely but exceptionally
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performed (i.e. genetically-based incapacity to convert β-carotene into retinol) and the best practice includes the use of fasting serum collected in plain or gel separator or anti-coagulated tubes (heparin, EDTA) (Greaves et al, 2014). In this sense, using serum and Li-heparin plasma, an acceptable degree of agreement has been reported for most serum carotenoids except possibly for minor components (i.e.
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zeaxanthin, α-cryptoxanthin) especially at the lower end of the concentrations distribution (OlmedillaAlonso et al, 2005). Thus, the type of specimen (i.e. serum, Li-heparin plasma) does not appear to be a matrices).
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relevant analytical constraint, at least in relative terms or for comparative purposes (i.e. different food
As a laboratory medicine best practice, C18 columns and UV/Vis detection are recommended for the analysis of vitamin A and β-carotene, although mass spectrometry is recommended as an alternative detection method (Greaves et al, 2014). Traditionally, reversed-phase HPLC on C18 columns have been widely used for separating carotenoids while polymeric C18 phases have provided an acceptable selectivity for similar carotenoids such as the cis- and trans- forms and structural isomers (i.e. lutein and zeaxanthin), specially under gradient elution conditions (Granado et al, 1991; Olmedilla et al, 1997). Nevertheless, non-endcapped columns with triacontyl (C30) ligands were developed specifically for a better isomer resolution (Sander e tal, 1994) and have become widely used for research purposes (Arathi et al, 2015; Gupta et al, 2015; Bohn et al, 2015). Morever, despite it usually requires longer run times, limiting its utility in large studies and clinical practice, rapid and sensitive methods have been also developed (Gupta et al, 2015). Alternatively, the development of columns with particles < 2 µm has provided new approaches to resolve carotenoid mixtures in much shorter times (ultra-fast HPLC),
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ACCEPTED MANUSCRIPT improving resolution, increasing sensitivity and reducing substantially the associated costs (Granado et al, 2010a). In addition, the occurrence of carotenoids cleavage products is crucial to assess their potential bioactivity. In this context, carotenoids in biological samples are frequently detected below nanomolar concentration and metabolites exist at picomolar concentration (Khachik et al,1997). Therefore, improvement in analytical techniques to resolve and identify retinyl esters and carotenoid metabolites under in vivo conditions (i.e. TRL) requires sensitive methods (i.e. HPLC coupled with MS/MS or
NMR) as well as the availability of appropriate standards (Kopec et al, 2013; Arathi et al, 2015; Gupta
3.2.
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et al, 2015; Bohn et al, 2015). Timing of intervention.
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In general, single dose studies aim to assess bioavailability, pharmacokinetic and acute effects while medium/long-term trials are intended to evaluate chronic (cumulative) exposure, functional changes or
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modifications of risk factors. The information obtained is somewhat different since it is interpreted in terms of postprandial response/metabolism (<48-72h), and repletion and/or saturation processes (> 1
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week), respectively (Granado-Lorencio & Olmedilla-Alonso, 2003).
Overall, a relationship exists among the type of marker (analytical or functional), the biological specimen needed and the time required for a change (FIGURE 1). Broadly, recent exposure may be
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assessed by analytical markers measured in biological matrices with relatively “fast turnover” (i.e. postprandial TRL fraction, serum, urine, feces, breath, breast milk, bile). On the contrary, for cumulative exposure (i.e. multiple dose studies), analytical markers measured in samples with “longer turnover” (i.e. lymphocytes, buccal mucosa cells, colonocytes) or storage tissues (i.e. adipose tissue)
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are preferable. Similarly, for functional tests, the markers will depend on the intervention time so that
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this should be long enough to provoke a change in the markers and matrix selected. Multiple-dose intervention studies may be useful for comparing population groups, food sources, ingredients or functional effects but this approach cannot be used to quantify the rate and extent of carotenoid absorption (Faulks & Southon, 2005). Moreover, as the time of intervention increases,
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changes in biomarkers may reflect better a cumulative effect (i.e. predictive value for a clinical outcome) although the potential confounding factors (i.e. tissue metabolism), the rate of drop-outs, and the costs increase as well. Additionally, biomarkers with a high methodological variability (i.e. subject-dependent
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carotenoid metabolites) often require a higher number of participants or repeated measures to account for within-person variations and to provide a more reliable index of exposure, status and response [Ellwood et al, 2014; Rhagavan et al, 2016]. 3.2.1.
Postprandial response, clearance and excretion
The preferred method of measuring carotenoid absorption and bioconversion is to isolate the triacylglycerol-rich lipoprotein plasma fraction (TRL) which contains newly absorbed carotenoids and formed retinoids (Van den Berg et al, 2000; Failla & Chitchumroonchokchai, 2005; Carbonell-Capella et al, 2014). However, it is necessary to discriminate between newly absorbed from endogenous carotenoids and to consider the clearance kinetics, since these factors may affect the estimated area under the curve (AUC) (Faulks & Southon, 2005). In this context, it is important to note that the use of large-doses and medium-/long-term supplementations may lead to non-physiological effects by altering the normal bioavailability, metabolism and clearance rates, or lead to non-linear responses (Welch et al,
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ACCEPTED MANUSCRIPT 2011). In addition, carotenoids (especially xanthophylls) may be transferred between lipoproteins and be taken up by extrahepatic tissues before reaching the liver and thus, analyses of this fraction may not provide an accurate estimate of the “true” relative absorption (van den Berg et al, 2000; Furr & Clarke, 2003). Plasma response (kinetics) of individual carotenoids also differs so that the timing of sample collection becomes essential. For example, in humans, the time for peak concentrations in TRL fractions was shorter for lutein (about 2h) than for β-carotene and lycopene (about 4-6 h) (O´Neill & Thurnham, 1998) while astaxanthin showed two peaks (at 7 and 16h) coinciding with peak plasma triglyceride concentrations (Furr & Clarke, 2003). Normally, after oral dose, peak concentrations of carotenoids in
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plasma occur at 6-48h (depending upon the dose and the frequency of measurements) which is slower than the time of appearance of cholesterol and triglycerides after a meal (usually 4-6h). Moreover, there is a second peak in plasma carotenoid which seems to accompany the later meal, making difficult to
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measure accurately the AUC (van den Berg et al, 2000; Faulks & Southon, 2005).
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Carotenoids also display distinct half-life in humans which may be related to differential deposition in target tissues, utilization or metabolism. It has been reported that all-trans-lycopene displays a shorter half-life than the cis- isomers (Moran et al, 2015). Similarly, compared to lycopene,
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the clearance rate of phytoene from chylomicrons was slower (Moran et al, 2016) while that of capsanthin appeared to be much faster (Oshima et al, 1997). Additionally, in the absence of carotenoid intake, plasma levels fall to 10-75% of baseline concentrations after 2-3 weeks, with a faster decline
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during the first 2-7 days (Rock et al, 1992). Importantly, many bioavailability studies include a “run-in” period (usually, a low/free carotenoid diet) to accommodate volunteers and ensure compliance. However, this “depletion” diet may lead to overestimate the “true” magnitude of the response by
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considering baseline concentrations as those after a clearance period. The routes of elimination may be also relevant when assessing carotenoid bioavailability.
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Several studies indicate that more than 70 % of the carotenoids may remain in the final digesta (Maiani et al, 2009; Bohn et al, 2015) and consistently, kinetic studies in man showed that the cumulative elimination of 14C in faeces was 51% of the administered 14C-β-carotene dose (Ho et al, 2007). In this sense, the presence in human faeces of free /esters forms and cis- /trans- isomer ratio reflecting the
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diet supports the use of faecal carotenoid profile as a suitable biomarker (and non-invasive approach) regarding responsiveness (i.e. compliance), nutrient stability and availability of chemical forms reaching the colon (Hernandez-Alvarez et al, 2015). However, although it is easy to collect fresh fecal samples,
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interpretation should be made cautiously since fecal carotenoids reflect different sources including unabsorbed dietary content, sloughed gastrointestinal cells, carotenoids excreted in bile and, possibly, microbiota-related metabolites. Finally, carotenoid excretion via the urine has long been considered to be very low. However, tracer studies have shown that 16–30% of orally administered 14C-β-carotene was excreted in urine within 72hours-21 days (Ho et al, 2007). Similarly, after a single microdose of 14C-lycopene, radioactivity was detected in exhaled carbon dioxide (3%) and urine (18%) within 4 h and 12 h, respectively (Ross et al, 2011). Thus, it is likely that urinary compounds represented oxidized and conjugated metabolites (Failla & Chitchumroonchokchai, 2005). Additionally, carotenoids are eliminated without modifications into bile (Leo et al, 1995), although the occurrence of a potential enterohepatic recirculation is unknown. Similarly, carotenoids in breast milk reflects serum profile (Khachick et al, 1997; Canfield et al, 2003) and constitutes another route of elimination.
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ACCEPTED MANUSCRIPT 3.3.
Biological Matrix.
Both serum and tissue carotenoid concentrations are used as biomarkers of recent and longterm exposure, respectively. Humans are yellow-fat animals and accumulate carotenoids reasonably well (i.e. adipose, liver and skin). For example, carotenoid intake correlates with adipose tissue concentrations of α-carotene, β-carotene, β-cryptoxanthin, cis-lycopene, and total carotenoids (Chung et al, 2009) while dietary carotenoids such as α-, γ-, β-carotene, lycopene, lutein, zeaxanthin and their isomers may be found in human skin (Wingerath et al, 1998). However, selective deposition of some carotenoids in certain tissues is well-known. Lycopene is
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concentrated in the prostate, testes or liver (Moran et al, 2013) while lutein and zeaxanthin are especially abundant in retina (macula) where they represent 80-90% of the total carotenoids (Handelman et al, 1992). The brain, like the retina, also appears to accumulate xanthophylls and
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correlate with serum carotenoids so that these have been proposed as biomarkers to predict brain carotenoid concentrations (Johnson et al 2011; Hammond, 2015). Similarly, concentrations of
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carotenoids in breast milk may be used as an index of maternal dietary intake (Canfield et al, 2003; Lipkie et al, 2015; Tanumihardjo et al, 2016) although they are associated with fat content, vary with the lactation stage and lutein and zeaxanthin levels are higher than those of β-carotene, in contrast to
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their concentrations in maternal plasma (Furr & Clarke 2003; Canfield et al, 2003). Importantly, when planning bioavailability studies in humans, ethical considerations are mandatory so that invasiveness for sample collection becomes essential. While adipose tissue and liver
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may be adequate matrices to assess long-term intake/exposition, other (non-invasive) tissue samples may be easily accessible (i.e. buccal mucosal cells, exfoliated colon cells) despite factors such as cell turnover must be considered. Alternatively, macular pigment optical density (measured by heterochromatic flicker photometry) and skin carotenoid status (assessed by reflectance based methods
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and Raman spectroscopy) may be used as markers of long-term dietary exposure. 4.- MARKERS OF CAROTENOID BIOAVAILABILITY 4.1.- Qualitative features
The influence of the diet on the profile and levels of carotenoids in serum has been known for
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decades and it is widely accepted that serum carotenoids represent good biomarkers of fruit and vegetable intake at ecological level (Olmedilla et al, 2001; Al-Delaimy et al, 2005; Maiani et al, 2009,
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Bohn et al, 2015).
Humans are considered indiscriminate carotenoid absorbers (Goodwin 1984), meaning that we are capable of absorbing a wide range of carotenes and xanthophylls. Approximately, 40–50 carotenoids are available in commonly consumed fruits and vegetables which can be divided into carotenoid epoxides, mono- and di-hydroxy-carotenoids, hydrocarbon carotenoids and carotenol acyl esters. From these, a significant proportion can be absorbed through the intestine, incorporated (unaltered) into chylomicrons and be available systemically. Broadly, bioavailability partly depends on the carotenoid structure (i.e. provitamin A capacity) and, in general, polar carotenoids are preferably incorporated into mixed micelles and tend to be of higher bioavailability, as may be the case for lutein vs β-carotene, free vs. esterified xanthophylls and cis- vs trans-forms (Bohn, 2008). However, up to∼34 carotenoids (including geometric isomers, metabolites and acyclic colorless carotenes) have been identified in human serum and tissues (Khachick et al, 1997) (TABLE 2). Nevertheless, just 6 of these, α-carotene, β-carotene, lutein,
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ACCEPTED MANUSCRIPT zeaxanthin, β-cryptoxanthin and lycopene comprise ∼90% of that total number (Bieri et al, 1985; Olmedilla et al, 2001). Epoxy-carotenoids Epoxy-carotenoids (i.e. neoxanthin, violaxanthin) are ingested together with lutein from green vegetables but it has been long considered not to be absorbed (possibly destroyed upon gastrointestinal conditions or rapid metabolism) due to their absence in serum and tissues (Khachick et al, 1991). Nevertheless, under in vitro conditions, free epoxy-carotenoids have been found in supernatants (Granado-Lorencio et al. 2007a) and Caco-2 cells were reported to take up neoxanthin and fucoxanthin
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(Sugawara et al. 2001).
In humans, 5, 6-epoxy-β-carotene may be absorbed as indicated by their presence in circulating
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blood (Barua et al, 1999) while epoxy-xanthophylls such as lutein 5,6-epoxide, violaxanthin and capsanthin-5, 6-epoxide, or any of their metabolites, were not detected after a single-dose (Barua and
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Olson 2001; Pérez-Gálvez et al, 2003; Kotake-Nara et al, 2011). Similarly, even after 1-week intake of epoxy-xanthophyll-rich diets, plasma concentrations of neoxanthin, its metabolites (neochrome stereoisomers) and fucoxanthinol (a gastrointestinal metabolite of fucoxanthin) remained very low low (Asai et a, 2004; Kotake-Nara et al, 2011).
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(about 1 nmol/l) or absent, suggesting that the plasma response to dietary epoxy-xanthophylls was very
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Keto, keto-hydroxy- and carboxylic carotenoids
Upon acute ingestion, keto and keto-hydroxy-carotenoids (i.e. astaxanthin, capsanthin, capsorubin, sapotexanthin, cryptocapsin) have been found in human chylomicrons and plasma (Oshima
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et al. 1997; Odeberg et al. 2003; Coral-Hinostroza et al. 2004; Chacón-Ordoñez et al, 2017). Astaxanthin and several radio-labeled metabolites were reported in humans reaching peak levels at 11.5
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h after administration (Kistler et al, 2002) while capsanthin and capsorubin (i.e. paprika oleoresin) appear to be less bioavailable compared to common carotenoids also present in the resin (Oshima et al, 1997; Etoh et al, 2000; Perez-Galvez et al, 2003). Also, upon supplementation, naturally occurring dicarboxylic apo-carotenoids i.e. bixin and its metabolite (norbixin), have been found in human blood,
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showing maximal concentrations at 2-4h (bixin) and 4h (norbixin) and a complete clearance by 8h and 24h (for bixin and norbixin, respectively) (Levy et al, 1997).
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Colourless carotenes and precursors Postprandial absorption of phytoene, phytofluene, ζ-carotene and neurosporene (i.e. chylomicrons) in humans appears to follow the same absorption time-course as lycopene and other carotenoids (Cooperstone et al, 2015). Additionally, both phytoene and phytofluene have been identified in breast milk and human tissues and appeared to be more bioavailable and more efficiently deposited in tissues compared to other carotenoids (i.e. lycopene) (Melendez-Martinez et al, 2015; Moran et al., 2016). Xanthophyll esters Foods containing xanthophyll esters (i.e. fruits) may transfer free xanthophylls more efficiently than those containing only free forms, a fact and that might explain partly the in vivo provitamin A value of β-cryptoxanthin-containing foods (Burri, 2015; Burri et al, 2016). However, the available data are inconsistent and suggest that the degree of bioavailability of free and ester forms of carotenoids may
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ACCEPTED MANUSCRIPT depend on the carotenoid and the matrix assessed. Thus, comparative studies showed an enhanced bioavailability for zeaxanthin dipalmitate compared to the non-esterified form (Breithaupt et al. 2004) while comparable bioavailability were observed for free and β-cryptoxanthin esters (Breithaupt et al, 2003). For lutein, a higher bioavailability of ester forms has been reported (Bowen et al, 2002) although no differences has been also found (Chung et al, 2004) and lutein response from supplements containing free lutein was greater than that from those containing lutein esters (Norkus et al, 2010). Under in vitro conditions, ester forms have been found in the aqueous-micellar phase and minor, although quantifiable, amounts have been reported to be taken up by Caco-2 cells (GranadoLorencio et al. 2007a; Failla & Chitchumroonchokchai, 2005). Thus, the postprandial absence of
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xanthophyll esters (i.e. lutein, β-cryptoxanthin and astaxanthin) in humans has led to the assumption that xanthophyll hydrolysis is a prerequisite for xanthophyll absorption (Wingerath et al, 1995; Breithauptet al. 2004; Schlattereret al. 2006; Granadoet al. 2010; Burri, 2016). Nevertheless, lack of
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sensitivity to detect low amounts of esters in TRL fractions and/or first-pass metabolism during absorption (i.e. hydrolysis at the enterocyte) may also explain their postprandial absence. Only upon
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long-term supplementation, the presence of xanthophyll esters in serum have been reported (Granado et al, 1998) although esters of lutein, zeaxanthin, anhydrolutein and α- and β-cryptoxanthin are
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normally present in human skin (Wingerath et al, 1998). Cis-isomers
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Most carotenoids in plants exist in the all-trans configuration although food processing (i.e. heating, canning) can induce cis-isomerization. However, the cis-trans ratio of carotenoids in chylomicrons and serum remains fairly constant, regardless of their proportion in the diet, the population studied or the isomer distribution in the supplements provided (Stahl et al. 1995; You et al.1996; Olmedilla et al.
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2002; Granado-Lorencio et al, 2007b).
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Both the cis-isomer profile and the cis/trans ratio vary in tissues as well. Apparently, isomerization of β-carotene did not occur in the gastrointestinal tract and both all-trans and 9-cis β-carotene are similarly absorbed (Faulks et al, 1997). However, after an oral dose containing all-trans and 9-cis-βcarotene, humans show a much higher level of all-trans-β-carotene than 9-cis-isomer in chylomicrons
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and very-low density lipoproteins (VLDL) (Stahl et al, 1995). In fasting conditions, 13-and 15-cis-βcarotene are found in serum at only 5-10% of the all-trans isomer while a higher proportion if cisisomers are found in tissues (10-40%) (Stahl et al, 1992; Van Vliet, 1996; Faulks & Southon, 2005). In
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addition, 9-cis-β-carotene has been reported in tissue samples but not in serum (Stahl et al, 1992). Isomeric composition of lycopene also differs in the diet, serum and human tissues. It is well known that all-trans-lycopene predominates in the main dietary sources of lycopene (i.e. tomatoes and tomato products), but plasma and tissues contain relatively greater concentrations of cis isomers. All-translycopene accounts for 95% in foods, about 65% in chylomicrons and 45-50% in serum with at least three cis-isomers (9-, 13-, and 15-cis) being present in serum. Although this pattern may suggest that cis-lycopene isomers may be more bioavailable (Bohn, 2008), studies in humans with C13-lycopene have revealed that post-absorptive trans-to-cis-lycopene isomerization, and not the differential bioavailability of isomers, drives tissue and plasma enrichment of cis-lycopene (Ross et al, 2011; Moran et al, 2015). On the contrary, it has been reported that astaxanthin cis-isomers were selectively absorbed into plasma, comprising ~32% of total astaxanthin postprandially (Coral-Hinostroza et al, 2004).
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ACCEPTED MANUSCRIPT Overall, although several mechanisms may promote a selective enrichment of cis-isomers of some carotenes and xanthophylls (i.e. cis-isomers are less likely to crystallize, preferentially micellarized or more readily taken up by intestinal cells) (Cooperstone et al, 2015), these apparently affect differently to each carotenoid (i.e. the predominance of all-trans-β-carotene vs cis-lycopene in serum). 4.1.1. In vivo metabolites as surrogates of carotenoid bioavailability Approximately, ~ 10% of dietary carotenoids can be converted into vitamin A and thus, postprandially, provitamin and non-provitamin A carotenoids as well as retinyl esters are incorporated into chylomicrons. During postprandial states, the measurement of retinyl esters (mostly retinyl
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palmitate and esters with other long-chain fatty acids) and carotenoids in TRL fractions has been frequently used to assess both the degree of absorption and conversion (AUC), at least in comparative terms. Under fasting conditions, however, retinol (bound to retinol binding protein and transthyretin) is
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the major circulating form and retinyl esters represent<10% of the total vitamin A in serum (Tanumiharjdo et al, 2016). Additionally, serum retinol levels are homeostatically regulated so that its
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use as a marker of bioefficacy is limited.
Dehydration products (i.e. anhydrolutein) have been reported in human serum and tissues and
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suggested to be non-enzymatically formed in the stomach under acidic conditions (Khachick et al, 1991, 1995, 1997). Other metabolites of lutein and zeaxanthin (i.e. 3′-Epilutein, meso-zeaxanthin, 3´oxolutein, 3 -methoxy-zeaxanthin) have also been reported in human serum, milk and ocular tissues
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(Khachik et al., 1995, 1997; Bernstein et al., 2001; Bhosale et al, 2007; Bhosale & Bernstein, 2005;Thürmann et al, 2005). Additionally, oxidative metabolites of lutein characterized as mono-keto, mono-hydroxy and di-keto-carotenoids have been also reported in human serum (Khachick y cols, 1991; 1995; 1997). Moreover, since these keto-carotenoids are not widely found in foods and increase in
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serum upon lutein supplementation, it is suggested that these carotenoids are formed in vivo (Khachik et al, 1997; Granado et al, 1998). Similarly, the presence and in vivo formation of lycopene epoxides
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(i.e. 2, 6-cyclolycopene-1,5-diols) in human tissues such as prostate, lung and colon have been also reported (Khachick et al, 1997; 2002). Thus, the occurrence of these metabolites in serum and tissues indicates that several carotenoids may undergo chemical modifications under in vivo conditions. Apo-carotenoids (i.e. β-apo-carotenoids and apo-lycopenoids) are present in the diet (i.e.
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processed mango juice, dried apricots, tomato extracts and paste) and can be formed via autoxidation, thermal degradation or during food processing (Eroglu et al, 2013). Nevertheless, it has been reported the presence of β-apo-carotenals and β-apo-carotenone in human and animal blood and tissues (Wang
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et al, 2015) and their formation under in vitro conditions as a result of lycopene and xanthophylls metabolism (Goupy et al 2012; Mein et al, 2011; Sharoni et al, 2012). In this context, excentric cleavage of ingested β-carotene occurs in vivo and the formation of several apo-carotenoids have been tentatively identified in humans (i.e.β-apo-8´-carotenal, β-apo-13-carotenone) (Ho et al, 2007; Eroglu et al, 2012). Moreover, the appearance of 14C- β-apo-8´-carotenal in plasma 3 days after dosing suggests that β-apo-carotenals are formed in peripheral tissues (Ho et al, 2007). Similarly, lycopene metabolites (lycopenoids) have been reported in serum and human milk (Lindshield et al. 2007) and several apo-lycopenals (i.e. 6´-, 8´-, 10´-, 12´- and 14´-) were found in human plasma of subjects after consuming tomato juice (also containing lycopenals), although whether the plasma apo-lycopenals originated from enzymatic cleavage of lycopene or from consumption of apo-lycopenals containing fruits and vegetables is not clear (Kopec et al.2010; Eroglu et al, 2013). Overall, despite different carotenoid metabolites (i.e. keto-carotenoids, cyclo-lycopene diols, apo-carotenoids) have been found in serum and tissues and may respond (increase) upon
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ACCEPTED MANUSCRIPT supplementation, they have not been frequently assessed so that their utility as biomarkers of bioavailability (exposure or effect) remains to be established. 4.2.- Quantitative considerations Many designs to assess carotenoid bioavailability yield information regarding relative bioavailability (compared to a reference dose or control) and thus, studies using different methodological approaches (i.e. food matrix, dose, timing) can lead to different results when determining fractional absorption (van den Berg et al, 2000; Granado-Lorencio & Olmedilla-Alonso, 2003; Failla &
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Chitchumroonchokchai, 2005). For carotenoids, both absorption of unaltered compounds (provitamin and non-provitamin A carotenoids) and conversion into retinol (first-pass metabolism) must be considered. Human studies
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performed in the 1960´s identified retinyl esters as the major metabolites of β-carotene in the lymphatic system (61-80% of labeled compounds), with variable amounts of unaltered β-carotene (up to 30%)
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(Goodman et al, 1966; Blomstrad et al, 1967). Using postprandial (TRL) models and a 1:2 molar conversion factor (β-carotene/retinol) percentages have been established between 35-71% (Van Vliet et al, 1996; Granado et al, 2001) while using labeled β-carotene and 1:1 molar ratio, about 20% of the
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absorbed β-carotene was converted into retinol (Novotny et al, 1995). More recently, the estimated βcarotene bioefficacy (a combination of both absorption and conversion) was estimated at 13.5%
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±6.02%, although a high inter-individual variation (CV: 44%) was also observed (Green et al, 2016). In contrast to serum retinol which is tightly (homeostatically) controlled, serum concentrations of carotenoids in humans are not regulated. Overall, the absorption efficacy of carotenoids is highly variable ranging between 5-80% (Parker, 1996; Erdman et al, 1993; De Pee & West, 1996). Assuming a
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dose-response effect, a proportional variation of serum /tissue levels of carotenoids with increasing amounts of intake is expected (i.e. “linearity”), at least at dietary achievable levels. In this sense,
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significant but highly variable correlations between dietary intake and serum concentrations have been reported (r= 0.1- 0.7) depending on the carotenoid and the study (Furr & Clark, 2003; Al-Delaimy et al, 2005). Consistently, carotenoid profile and levels in tissues (i.e. adipose, prostate, liver, colon cells) seems to respond to nutritional interventions although the magnitude of the associations differ for
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various tissues (Maiani et al, 2009; Sen et al, 2013; Bohn et al, 2015). Factors affecting the magnitude of the correlations are both methodological (i.e. dietary
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assessment methods, food carotenoid databases) and physiological (i.e. first-pass metabolism, tissue deposition). Alternatively, due to the lack of robust linear relationships, (semi)quantitative relationships have been approached by classifying subjects according to their level of exposure (i.e. quartiles of intake and serum levels) so that an acceptable degree of agreement (correct classification) may be obtained. Even so, the concordance between intake level and serum concentrations should be interpreted cautiously since they do not necessarily reflect the relative amount of structural and geometrical isomers. For example, levels of lycopene in prostate correlated with post-intervention plasma lycopene concentrations (r 0·60, P =0·001) but plasma and prostate cis-isomers accounted for 47 and 80 %, respectively (Grainger et al, 2015). Similarly, in serum, the lutein: zeaxanthin ratio is about 3:1 whereas in macula, this ratio reaches values of up to 1:2. Nevertheless, concentrations of lutein and zeaxanthin in serum and tissues (i.e., macula) increase on consuming lutein-rich foods or supplements. Moreover, this increment parallels the increase in MPOD (macular pigment optical density) and the improvement in visual function (Trieschman et al, 2007; Schalch et al, 2007; Olmedilla et al, 2003), demonstrating
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ACCEPTED MANUSCRIPT consistently a relationship between serum and tissue markers (lutein levels, MPOD) and functional indicators (i.e. visual function) (Olmedilla et al, 2003; 2014). Compared to drugs, however, the (functional) effect derived from nutritional interventions is expected to be small although markers of exposure (i.e. serum carotenoids) may change significantly. Due to the endogenous presence of carotenoids, large doses have been frequently used to provoke a measurable response. However, this approach has been criticized as they may alter the physiological response and assume non-saturable models and linear dose-responses. Additionally, effects upon intervention may be related to nutritional status, the dose and the time of the intervention, all of which may influence the response and even lead to a “ceiling effect” (Olmedilla et al. 2002; Schlatterer et al.
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2006). Moreover, postprandial behaviour does not predict long-term effects, and acute (single dose) responses usually show larger inter-individual variability than chronic (multiple) intakes (Borel et al. 1998; Olmedilla et al. 2002; Granado-Lorencio & Olmedilla-Alonso 2003; Faulks & Southon, 2005). For
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example, using a beverage enriched in lutein at two levels, a greater postprandial AUC was obtained with the high dose although the percentage of absorption was not different when standardized to the
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amount supplied. Nevertheless, upon regular consumption, serum increments at 7 and 14 days were statistically higher on consuming the high-dose beverage (Granado-Lorencio et al, 2010b). Thus,
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comparison of results from different protocols should be interpreted cautiously. Multiple-dose supplementation studies are also used to assess the bioavailability (i.e. bioactivity or functional effects) of carotenoids in humans so that the linearity of the response becomes an
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important issue. Using lutein, lycopene, α-+ β-carotene, astaxanthin and capsanthin, multiple-dose human studies have reported the presence of a “plateau effect” in serum concentrations of carotenoids (Oshima et al, 1997; Olmedilla et al, 2002; Meinke et al, 2010). Thus, regardless of the underlying mechanisms (i.e. dose, duration of the study, type of biomarker, number of measurements and
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physiological mechanisms), the loss of linearity in the response questions the reliability and utility of serum levels as markers of exposure (bioavailability) above certain concentration threshold. On the
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contrary, consistent with a longer half-life of carotenoids in tissues, carotenoids appear to decrease much more rapid in serum than in tissues (i.e. skin) (Meinke et al, 2010) and thus, levels of carotenoids in tissues could provide, a priori, better linearity and reflect more successfully the cumulative exposure and effects. However, again, the potential presence of carotenoid metabolism in tissues and the cell
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turnover should be also considered.
Overall, in biological systems, large deviations from linearity may occur and, in terms of efficacy
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(function), linear relationships are the exception rather than the rule (Faulks & Southon, 2005). Obviously, when linearity is lacking, the utility of the marker is lost since exposition cannot be reliably related to the effect. Moreover, in many studies, biological samples to measure both analytical and functional markers are usually collected at baseline and at the end of the intervention. Unfortunately, under these conditions, it is not possible to assess when the changes occur, if a minimum amount of the bioactive is needed to get an effect (threshold effect), if a (analytical or functional) change is linear over time, if a saturation (“plateau”) effect is present or for how long the effect is maintained upon discontinuation. 4.3.- Markers of overexposure While clinical signs and biochemical markers of vitamin A toxicity (i.e. increased retinyl esters in serum) exist, this approach is not applicable to carotenoids. Traditionally, dietary carotenoids are considered to be safe and free of secondary effects beyond the potential, benign and reversible
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ACCEPTED MANUSCRIPT yellowish-orange discoloration of the skin (i.e. palms, soles, naso-labial folds) usually referred to as carotenodermia (xanthosis, xanthoderma or pseudoicterus). It is well known that infants are especially prone to develop carotenodermia due to their dietary intake as well as patients taking β-carotene supplements (i.e. erythropoyetic protoporphyria). However, carotenoid-related skin pigmentation may be also observed in subjects with high intake of carotenoidrich foods (i.e. mango, oranges, carrots, tomato products) and supplements. Noteworthy, carotenodermia appears to develop differently according to the carotenoid provided. Upon long-term supplementation in apparently healthy volunteers, carotenodermia was observed in 95% of subjects taking α- + β-carotene, in 40%of those consuming lutein esters and in 25% of the volunteers taking
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lycopene supplements. These findings suggested that carotenoids were not equally deposited in the skin or that other tissues could be primary targets. Importantly, no changes in biochemical or haematological
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indices were associated with this clinical sign (Olmedilla et al, 2002).
In subjects taking tanning pills, it is recognized the potential occurrence of canthaxanthin
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retinopathy, a condition that may lead to long-standing visual changes including significant visual loss (Beaulieu et al, 2013). Upon discontinuation, subjects may be asymptomatic without functional defects but complete disappearance of the golden particles near the macular region could take up to 20 years
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(Hueber et al, 2011). Retinal paramacular cristal deposition and yellow-brown discoloration of the anterior lens capsule have been also reported in subjects with hypercarotenemia (i.e. serum β-carotene 75-325 µg/dl) and carotenodermia (Rahmani et al, 2003), and corneal rings may persist three years
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after the original presentation despite the serum carotene normalizes (Chang et al, 2012). More recently, corneal rings have been reported in four patients receiving vitamin supplementation for agerelated macular degeneration (i.e. AREDS 2 Study) (Eller et al, 2012). These patients had yellow peripheral corneal rings and a subtle yellowing of the skin (carotenodermia). Interestingly, serum
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carotene levels were normal in two of the three cases (<50 µg/dl) in which it was measured (Eller et al,
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2012), suggesting that serum levels may not reflect nor predict reliably the situation in certain tissues. Carotenodermia is usually associated with high levels of carotenoids in blood, a condition referred to as hypercarotenemia (>300 µg carotenoids/dl) (Underwood, 1984). Hypercarotenemia, however, is not a cause of disease but it may be present in some clinical conditions (hypothyroidism,
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diabetes mellitus, renal or liver disease, anorexia nervosa, brain tumor) (Rock & Swenseid, 1993; Olmedilla et al, 1995) and when an incapacity to convert β-carotene into retinol exists (Sharvill, 1970; Lindquist et al, 2007; Maruani et al, 2010). Additionally, the presence of xanthophyll esters in serum has
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been reported in subjects consuming lutein supplements and displaying serum lutein concentrations> 1.05 mmol/l (Granado et al, 1998). Although ester forms represented less than 3% of lutein levels in serum, they were present after one month of supplementation and, interestingly, before the occurrence of carotenodermia.
Overall, however, based on the available evidence, the cutoff point of serum carotenoids above which carotenodermia and corneal rings appear, or the risk increases, cannot be established although it seems to vary for each carotenoid and probably be subject-dependent. 5.
MODULATING FACTORS OF MARKERS OF EXPOSURE AND RESPONSE In general, besides exogenous (food-related) factors, host-related and methodological issues
may influence analytical and functional markers of bioavailability (TABLE 3). Morever, although
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ACCEPTED MANUSCRIPT carotenoids may share chemical structure and properties, these factors may not equally affect the different subclasses of carotenoids. Physiological factors Results from bioavailability studies (i.e. status and response) may be influenced by a variety of host factors including nutritional status (Failla & Chitchumroonchokchai, 2005). In fact, the body can down-regulate bioconversion of provitamin A carotenoids so that high intakes of fruit and vegetables do not usually cause concern for hypervitaminosis A (Tanumihardjo et al, 2016). Also, since carotenoids are transported by lipoproteins and these are affected by several factors, it has been suggested that plasma
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carotenoids status and response should be lipid-standardized (Furr & Clark, 2003). Sex differences in serum levels of provitamin A carotenoids have been reported in several, but not all, populations (Furr & Clark, 2003; Olmedilla et al, 2001) and differential age-dependent responses
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have been observed in human intervention trials (Maiani et al, 2009). However, a decreased chylomicron response of lycopene, but not lutein and β-carotene, was found in older compared to younger subjects
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(Cardinault et al, 2003). Also, body mass index (BMI) and the conversion efficiency of β-carotene into retinol has been correlated so that subjects with more body fat showed a lower capacity of conversion
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(Tang et al, 2010).
Clinical conditions may also affect the bioavailability (status and response) of carotenoids. Hypochlorhydria, achlorhydria and acidic pH diminished the solubility and absorption (AUC) of β-carotene
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while syndromes responsible for fat malabsorption (i.e.cholestasis, pancreatic insufficiency, biliary cirrhosis, cystic fibrosis) are expected to decrease carotenoid bioavailability as well. Other clinical conditions such as type 1 diabetes mellitus has been associated with lower serum retinol but higher
Inflammation
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efficiency (Granado et al, 2001).
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provitamin A carotenoid status, a situation that is not apparently due to an impaired conversion
Nutritional studies are usually performed in control subjects (“apparently healthy”) but the bioavailability of carotenoids (i.e. response) under subclinical conditions (i.e. chronic inflammation) may differ. In general, infection and inflammation may cause an increased demand (intake), and may alter
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metabolism or excretion of nutrients, reducing the availability and transference to tissues (Duncan et al, 2012; Brenashan & Tanumihardjo, 2014). During the acute phase response, more than 200 plasma proteins are modulated of which
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approximately 50% are involved in nutrient transport or regulation of nutrient status (Raiten et al, 2015). Overall, inflammation affects different nutrient biomarkers including retinol and carotenoids. Plasma retinol concentrations may decrease by as much as 50% during infections and systemic inflammation (Duncan et al, 2012;Thurnham & Northrow-Clewes, 2016) while plasma carotenoids may fall rapidly in states with fever and recover when fever declines (i.e. children with pneumonia) (Goodwin, 1984). Additionally, concomitant infection with intestinal helminthes, Helicobacter pylory or other microorganisms may impair carotenoid absorption or utilization (Failla & Chitchumroonchokchai, 2005; Maiani et al, 2009). However, there is not necessarily a linear relationship between nutrient biomarkers and acute response indicators (i. e. C-reactive protein) and thus, on assessing both status and response (i.e. bioavailability), the presence of subclinical chronic inflammation or acute phase response should be considered and, if necessary, adjust the results to provide a reliable measurement (Thurnham & Northrow-Clewes, 2016).
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ACCEPTED MANUSCRIPT Dietary factors and interactions It is well-known that bioavailability of dietary carotenoids is influenced by several dietary factors (De Pee & West, 1996) and a critical step is their liberation from the matrix and its solubilisation into mixed micelles. Natural deposition of carotenoids (i.e. solid-crystalline aggregates, lipid-dissolved forms) in plant and animal-based foods has been shown to be critical on the liberation efficiency (in vitro and in vivo) and thus, on the bioavailability of carotenoids (Schweiggert et al, 2012; 2015). Moreover, ripening-induced changes in the deposition state of carotenoids (i.e. protein-bound in chloroplasts to tubular chromoplasts) may also have a major impact by increasing in vitro bioaccesibility of xanthophylls esters (Hempel et al, 2017).
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It is also known that dietary fat generally increases carotenoid bioavailability and both the amount and the type of fat may affect the bioavailability of some (i.e. lutein), but not all, carotenoids (Roodenburg et al. 2000; Bohn 2008). Very low-fat diets can also decrease carotenoid digestion and
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absorption although β-cryptoxanthin may be less affected than others carotenoids (Burri et al, 2016). Similarly, long chain triglycerides (LCT), compared to medium chain triglycerides (MCT), increase
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chylomicron secretion and may enhance carotenoid absorption. Also, dietary fats/oils rich in unsaturated fatty acids appear to promote carotenoid in vitro bioaccesibility (i.e. β-carotene, lycopene) by enhancing their micellarization during digestion and intestinal transport (Failla et al, 2014), although
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dietary fats rich in saturated fatty acids led to a higher bioavailability of lutein and zeaxanthin, as compared with fats rich in mono- and poly-unsaturated fatty acids (Gleize et al, 2013).
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The presence of absorption modifiers (i.e. metals, fiber), at dietary and supplementation levels, may also affect the bioavailability of carotenoids. Both in vitro and in vivo studies suggest that the degree of xanthophyll esters hydrolysis, the transference of free xanthophylls into micellar phase and dose-adjusted changes of i.e. β-cryptoxanthin may be higher in the presence of iron and milk (Granado-
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Lorencio et al, 2009). Consistently, supplementation with iron, zinc or both have been shown to increase the bioavailability of provitamin A carotenoids from foods, as measured by chylomicron response and
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conversion into retinol (Kana-Sop et al, 2015). On the other hand, plant sterols and water-soluble fibers (i.e. pectin) decrease the absorption of β-carotene, lycopene and lutein (Failla & Chitchumroonchokchai, 2005; Maiani et al, 2009; Bohn et al, 2015). However, although phytosterol intake at the recommended doses (i.e.2 g/day) may lower serum levels of carotenoids (Bañuls et al, 2010), this inhibitory effect may
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be eliminated if co-consumed (Granado-Lorencio et al, 2011). Carotenoid interactions appear to be present between carotenes and xanthophylls and among
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compounds of the same subclasses as they share common mechanisms at different levels (i.e. micellarization, affinity to transporters, provitamin A cleavage, exchange between lipoproteins). Much of the evidence suggests an interaction between ß-carotene and oxy-carotenoids such as canthaxanthin and lutein, and between ß-carotene and lycopene although the magnitude and the direction of the interactions may differ (van den Berg et al, 2000; Reboul et al, 2007; Maiani et al, 2009). Nevertheless, the impact of these interactions will also depend on the relative amounts of carotenoids, the matrix (vehicle) and the protocol used (i.e. single vs multiple dose). Nutrients and drugs share common pathways and mechanisms so that, at least theoretically, acute and chronic medications (i.e. omeprazole, ezetimibe) may alter absorption, serum status, distribution, clearance and metabolism of carotenoids, and viceversa (Boullata et al, 2012). However, there is a lack of information regarding the impact of common drugs on the bioavailability and metabolism of carotenoids. Noteworthy, carotenoids can also influence the pharmacological activity of drugs by modifying their absorption and metabolizing enzyme systems. Thus, lycopene may inhibit
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ACCEPTED MANUSCRIPT CYP1A1 and CYP1B1 and increase hepatic cytochrome P4502E1 (Rodriguez-Fragoso et al, 2011). Moreover, from a clinical perspective, some interactions may be beneficial. For example, β-cryptoxanthin may interact with certain drugs acting as an enhancer and reducing the toxicity of anticancer treatments (i.e. oxaliplatin) (San Millan et al, 2015). Genetic variability. On the basis of plasma response (absorption efficiency), human volunteers were classified as responders and non-responders (“poor” and “good” absorbers) (Failla & Chitchumroonchokchai, 2005). Discrimination of subjects on the basis of plasma response has been criticized since the lack of an acute plasma response does not necessarily indicate lack of absorption, as reported in ileostomy patients
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(Faulks et al, 1997).
Both serum carotenoid status and response to supplementation have been related to genetic
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differences (single nucleotide polymorphisms (SNPs)) of several genes involved in absorption and transport of carotenoids (i.e. SR-B1, CD-36, ABC G5/G8, NPC1L1)(Lietz & Hesketh, 2009; von Lintig et
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al, 2010;Herron et al, 2006; Lietz et al, 2012; Borel et al,2012; 2014; 2015a, b). Also, SNPs in the BCMO-1 gene (encoding β-carotene 15, 15´-oxygenase) have been associated with fasting β-carotene concentrations and the ability of this enzyme to convert β-carotene into vitamin A (Leung et al, 2009)
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and SNPs in BCMO-1 and CD36 genes were associated with circulating levels of lutein, uptake of carotenoids at target tissues and the MPOD (macular pigment optical density) response to lutein supplementation (Feigl et al, 2014). Similarly, tissue concentrations of lycopene are likely dictated by
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expression and genetic variation of lipoprotein receptors, transporters and metabolizing enzymes, thus influencing lycopene accumulation at targets sites of action (Moran et al, 2013). Overall, the available evidence supports a relevant role for SNPs of carotenoid transporters and
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metabolizing enzymes as determinants of circulating levels, tissue concentrations and response,
Gut Microbiota
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although the effect may differ according to the carotenoid supplied (Wang et al, 2013).
Unabsorbed carotenoids may be active in the gastrointestinal tract as they may be also
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metabolized during intestinal transit and exert biological actions. Interestingly, animal and human studies have shown that some dietary bioactives (i.e. β-cryptoxanthin) may be absorbed through the colon where they or their metabolites (i.e. apo-carotenoids) may exert biological activity (La Frano et al,
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2013). Nevertheless, although in vitro studies suggest that at least some carotenoids (i.e. β-carotene isomers, β-cryptoxanthin esters) may be stable upon simulated fermentation conditions (Mosele et al, 2016;Hernández-Alvarez et al, 2015), the effect of microbiota and the colonic absorption remain as a major gap in the knowledge of carotenoid bioavailability (Bohn et al, 2015). 6.- CONCLUDING REMARKS. Markers of exposure and response constitute essential tools to assess bioavailability of carotenoids. Overall, both concentrations of carotenoids in different matrices and functional markers (associated biological effect) may be used both in single-dose and long-term supplementation assays. However, in general, a relationship exists between the type of marker (analytical or functional), the biological specimen needed and the time of intervention required to detect changes. Analysis of markers in matrices with rapid turnover (i.e. TRL, serum) usually reflect short-term bioavailability while cumulative exposure may be better approached by measuring carotenoids in tissues and using functional
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ACCEPTED MANUSCRIPT markers (i.e. biological effect). In any case, the validity of the studies may be compromised by methodological and biological factors including the analytical uncertainties, the timing of sample collection, pre- and post-absorptive changes, the linearity of the response and in vivo metabolism. Thus, an integrated approach including the use of combined protocols (i.e. single and multiple dose), multiple biomarkers (i.e. trans- and cis-isomers, metabolites) and biological matrices (i.e. short- and long-term turnover) may provide a more reliable estimation of carotenoid bioavailability. In addition, the potential impact of genetic variability, physiological state (i.e. subclinical inflammation), the use of drugs and gut microbiota should be also considered on assessing and interpreting the bioavailability of carotenoids in
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humans.
Conflict of Interest; None declared.
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Figure legends
Figure 1.- Integrative scheme illustrating the relationships between study design, timing of sampling, biological matrix, type of biomarkers and influencing factors. This figure aims to show the relevance of combining the objective of the study, the type of intervention (short or long-term) and the
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appropriate biological matrix to measure changes in (bio) markers. Overall, short-term exposure may be approached by determining changes in analytical markers in “fast” turnover biofluids (i.e. serum, feces)
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while long-term exposure may be better evaluated by using functional tests and analytical markers in “low” turnover tissues. In both cases, interpretation of changes should be performed with caution since confounding (i.e. rate of absorption, tissue metabolism) and modulating factors (i.e. selective tissue uptake) may bias a direct relationship between markers of exposure and biological effects as surrogates
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of carotenoid bioavailability.
Table 1.- Examples of biological and health endpoints and functional (indirect) markers
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used as surrogates of carotenoid bioavailability (i.e. bioactivity). Biological end-points
Assessment Methods/ Tests
Antioxidant capacity
HPLC, ELISA, Comet assay, antioxidant enzymes activity,
(i.e. DNA damage and repair
TBARS, MDA and other lipid metabolites
Lipid /protein oxidation products) Metabolic changes and physiological changes
Insulin resistance/ glucose tolerance, lipid profile, metabolomic profile, P450 system activity, blood pressure, pulmonary function test, brachial-ankle pulse wave velocity, inflammation markers
Eye health
Dark adaptation, visual acuity, contrast sensitivity, retinal sensitivity
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Bone health
Bone mineral density, biomechanical tests, bone remodeling markers
Immune function
Cell (subpopulations) count, cytokines (IL-2, IL-1b, IFNg, TNF-a)
Gene expression/ Epigenetic changes
Up- / down regulation, -Omics (i.e. proteomic, metabolomics), Hypo- / hyper DNA methylation Microbiota profile (dysbiosis) and metabolite changes,
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Gastrointestinal health
fecal pH, variation in markers of intestinal permeability
Cognitive, behavioral and psychological function
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and inflammation
Mental performance (i.e. timed calculation,
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concentration), neuropsychological tests (i.e. 15 words learning test), cognitive performance (i.e. memory,
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psychomotor speed), behavioural tests Physical performance
Strength, endurance, muscle performance, aerobic
Skin health
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capacity, bicycle ergometer Skin color (yellowness, redness and luminance),
(i.e. beauty from within, anti-ageing)
prevention/inhibition of UVA/UVB induced oxidative
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stress and gene expression Infection challenge tests, exercise stress, glucose tolerance tests, stressor tests (i.e. mental & physical fatigue, skin irritation test)
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Table 2.- Summary of chemical forms of carotenoids potentially useful as markers for assessing short- and long-term bioavailability (i.e. exposure). In vitro systems Human Refs Species and molecular forms
Duodenal/Micellar / Caco-2
Post-prandial (TRL, Chylomicron)1
Plasma-Serum
Tissues (including breast milk)
Trans carotenes (i.e. β-carotene, lycopene, γcarotene, )
YES
YES
YES
YES
Colourless carotenes: (i.e. phytoene, phytofluene)
YES
YES
YES
CaroteneEpoxides (i.e. 5,6-epoxy-β-carotene)
NR2
Free xanthophylls (i.e. lutein, zeaxanthin, βcryptoxanthin)
YES
YES
Xanthophyll Epoxides (i.e. neoxanthin, violaxanthin, fucoxanthin, lutein-5, 6-epoxide, capsanthin5, 6-epoxide)
YES
NO
Keto-carotenoids (i.e. canthaxanthin) Hydroxi-keto-carotenoids (i.e. astaxanthin) Capsanthin, capsorubin (including cis-isomers and metabolites) Carboxi-carotenoid (i.e. bixin, norbixin) Xanthophyll esters (i.e. lutein, α- and βcryptoxanthin or astaxanthin)
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Khachick et al, 1997; 2006 Lipkie et al, 2015
YES
Khachick et al, 1997; 2006; Moran et al, 2016; Cooperstone et al, 2015
NR
Barua et al, 1999 Meléndez-Martinez et al, 2015
YES
Khachick et al, 1997; 2006 Lipkie et al, 2015
NO (i.e. lutein-5, 6-epoxide, capsanthin-5, 6-epoxide, violaxanthin) Very low (neoxanthin, fucoxanthin)
NR
YES
YES
YES (i.e.retina)
Barua& Olson, 1991; Failla & Chitchumroonchokchai, 2005; Pérez-Gálvez et al, 2003; Granado et al, 2007; 2011; Asai et al, 2008 Sugawara et al, 2001 Kotake-Nara et al, 2011 Kistler et al, 2002; Polara-Cabrera et al, 2010
YES
YES
NR
Oshima et al, 1997; Etoh et al, 2000 Pérez-Gálvez et al, 2003
NR
YES
YES
NR
Levy et al, 1997 Polara-Cabrera et al, 2010
YES
NO
NO (Except in lutein supplemented subjects)
YES (i.e. skin)
Wingerath et al, 1995; 1998; Granado et al, 1998; Barua & Olson 2001; Failla & Chitchumroonchokchai, 2005; Breithaup et al, 2004;
NR
YES (human homonegates) NR
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YES
U N YES
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Coral-Hinostroza et al, 2004 Schlatterer et al, 2006; Burri et al, 2016 Granado et al, 2007; 2010; 2011 Cis-isomers (i.e. cis-lutein, cis-β-carotene, cis-lycopene, cis-capsanthin, cis-astaxanthin)
YES
YES (relative proportion is carotenoid-dependent)
YES (relative proportion is carotenoid-dependent)
NR
NR
Apo-carotenoids (β-apo-13-carotenone, β-apocaroten-ols & β-apo-carotenals) Lycopenoids (Apo-lycopenals) Free retinol Retinyl esters
YES
N A
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2, 6-cyclo-lycopene-diols
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Metabolites3 Meso-zeaxanthin, keto- and keto-hydroxycarotenoids (i.e. ε,ε-carotene-3-3´-dione, 3-OH-β,ε-carotene-one, 3´OHε,ε-carotene-3-one) anhydrolutein(s) 3-epi-lutein(s) capsanthone (in vivo oxidized capsanthin)
YES (Pattern tissue-specific & carotenoid dependent)
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YES (i.e. breast milk, ocular tissues) YES
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YES
Not reported
YES
NR
YES
NR
-
NO
YES (RBP) (homeostatic control)
YES (low/ very low)
-
YES
YES (<5-10% in healthy, fasted subjects)
YES (storage form)
YES
E C
(human homonegates)
C A NR
YES (i.e. breast milk) Not reported
Khachick et al, 1997; 2006 Lipkie et al, 2015 Moran et al, 2015; 2016; Ross et al, 2011 Etoh et al, 2004
Khachick et al, 1997; 2006 Granado et al, 1998; Bhosale et al, 2005; 2007 Thürmann et al, 2005 Etoh et al, 2004 Schlatterer et al, 2006
Khachick et al, 1997; 2006 Tang et al, 1991; Wang et al, 1992; 2015; Ho et al, 2007; Eroglu et al, 2012 Kopec et al, 2010; Goupy et al, 2012; Mein et al, 2011; Sharoni et al, 2011
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1
Detected during post-prandial period (usually 2-8h). Some studies report detection in plasma/blood during this time after dose/meal rather than prepare triglyceride-rich lipoprotein (TRL) or chylomicron fraction specifically. Some carotenoids (i.e. xanthophylls) may be transferred to other lipoproteins or tissues during this period and thus, it is assumed they are from dietary origin. 2NR; Not reported, not assessed or below detection limit of the technique employed. 3
Some compounds referred to as metabolites are also present in foods although its major contributor is thought to be in vivo metabolism.
T P
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C S U
N A
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T P E
C C
A
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Pharmaco-kinetic vs cumulative exposure (i.e. single vs multiple dose) Dose and duration of intervention Timing of blood collection Linearity of response / Saturation (“ceiling” effect) Compliance Type of sample (short- vs long-term exposure/ effect)
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Biological matrix
Biological variation (i.e. circadian, seasonal)
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Cell turn-over (i.e. colon and buccal mucosal cells) Pre-, analytical and post-analytical factors
Stability during sample collection, transport and storage
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Resolution and detection limit
Lack of standards (i.e. metabolites) Between methods and between labs variability (Lack of method
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standardization) *
Widely accepted references ranges* Cutoffs with clinical or epidemiological relevance *
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Nutrient synergism (Lack of selectivity for causal links) *
Host-related factors
Genetic variability Affecting “static” markers/response
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Affecting “functional” markers/response
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SNPs of absorption, transport and metabolism systems SNPs for enzymes and processes involved in physiological function/effects DNA methylation, up- and down-regulation
Physiological factors
Age, gender and body mass index
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Epigenetic changes /nutrigenomic actions**
Gastric pH, biliar and pancreatic secretions Intestinal absorption limits (saturation thresholds) First-pass metabolism Differential transport, transference and tissue deposition Clearance rate Tissue-specific metabolism Vitamin A status and homeostatic control Malabsorption-related disorders (i.e. diarrhea, parasites) Physiological state (i.e. inflammation) Metabolism by microbiota Diet and drug-interactions Enterohepatic recirculation?
Lifestyle factors
Tobacco, alcohol, exercise
*Factors especially relevant for functional markers /tests. ** Also dependent from other nutrients such as folate, B12, vitamin K and A& methyl group donors.
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Graphical abstract
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ACCEPTED MANUSCRIPT Highlights
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Both host-related and methodological factors may influence static and functional markers
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Biomarkers constitute an essential tool to assess the bioavailability of carotenoids in humans. Markers for carotenoid bioavailability can be divided into direct and functional indicators. A relationship exists between the type of marker, the biological specimen and the time required for a change. Overdosing and long-term protocols may lead to saturation effects and the loss of linearity.
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F. Granado-Lorencio, I. Blanco-Navarro, B. Pérez-Sacristán PII: DOI: Reference:
S0963-9969(17)30138-2 doi: 10.1016/j.foodres.2017.03.036 FRIN 6643
To appear in:
Food Research International
Received date: Revised date: Accepted date:
19 November 2016 15 March 2017 19 March 2017
Please cite this article as: F. Granado-Lorencio, I. Blanco-Navarro, B. Pérez-Sacristán , Biomarkers of carotenoid bioavailability. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Frin(2017), doi: 10.1016/j.foodres.2017.03.036
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ACCEPTED MANUSCRIPT Biomarkers of carotenoid bioavailability Granado-Lorencio, F1 2,3*; Blanco-Navarro, I1,2,3; Pérez-Sacristán, B 1
Grupo Metabolismo y Nutrición, IDIPHIM;
2
1,2
Unidad de Vitaminas; 3 Servicio de Bioquímica Clínica,
Hospital Universitario Puerta de Hierro-Majadahonda, 28222 Madrid, Spain.
* Corresponding author: F. Granado-Lorencio
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Unidad de Vitaminas. Servicio de Bioquímica Clínica.
Hospital Universitario Puerta de Hierro-Majadahonda. 28222-Madrid (Spain) Telf: +34-911917578 / 7756
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Fax: +34-911916806
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e-mail: [email protected]
Abstract
The use of biomarkers constitutes an essential tool to assess the bioavailability of carotenoids in
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humans. The present article aims to review several methodological, host-related and modulating factors relevant on assessing and interpreting carotenoid bioavailability. Markers for carotenoid bioavailability can be broadly divided into direct, biochemical or “analytical” markers and indirect, physiological or “functional” indicators. Analytical markers usually refer to biochemical indicators of intake and/or status
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(short and long term exposure) while functional measures may be interpreted in terms of cumulative exposure, biological effect (bioactivity) or modification of risk factors. Both types of markers display
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advantages and limitations but, in general, a relationship exists among the type of marker, the biological specimen needed and the time required for a change. Humans may absorb a wide range of carotenes and xanthophylls and many of them may be found in serum and tissues. However, under physiological conditions, the several classes of dietary carotenoids may behave unequally leading to a different
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systemic profile and, moreover, they can be selectively accumulated at target tissues. In addition, some carotenoids may be chemically and enzymatically modified generating different oxidative metabolites and apocarotenoids. Quantitatively, the biological response upon carotenoid intervention (assessed by
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analytical and functional markers) is highly variable but the use of large doses and long-term protocols may lead to saturation effects and the loss of linearity in the response. Also, despite carotenoid exposition is considered to be safe, markers of overexposure include clinical signs (i.e. carotenodermia, corneal rings and retinopathy) and biochemical indicators (hypercarotenemia, xanthophyll esters). Overall, both host-related and methodological factors may influence analytical and functional markers to assess carotenoid bioavailability although the different subclasses of carotenoids may not be equally affected.
Keywords; Bioavailability, biomarkers, carotenoids, human study
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ACCEPTED MANUSCRIPT Index 1.- Introduction. 2.- Biomarkers 2.1.- Biomarkers of exposure 2.2. Functional biomarkers 3.- Methodological issues 3.1. Analytical points 3.2. Timing of intervention
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3.2.1. Post-prandial response, clearance and excretion 3.3. Biological matrix 4.- Markers of carotenoid bioavailability
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4.1.- Qualitative features
4.1.1.- In vivo metabolites as surrogate of carotenoid bioavailability
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4.2.- Quantitative considerations 4.3.- Markers of overexposure
5.- Modulating factors of markers of exposure and response
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6.- Concluding remarks
1.
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7.- References
INTRODUCTION
Carotenoids are fat-soluble pigments of plant origin that humans cannot synthesize although they can partially modify them. From a nutritional and physiological viewpoint, interest in carotenoids
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has been focused on the provitamin A activity of some of them but carotenoids also display other
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biological functions that may confer beneficial effects against chronic diseases. Prior to exerting their bioactivity, however, these compounds must be bioavailable. Bioavailability can be defined as the proportion of a dietary nutrient (or its metabolites) that is ultimately available for utilization or storage by target tissues after digestion, absorption and distribution (Ball, 1998).
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Nevertheless, because of the practical and ethical difficulties when measuring bioactivity and because of nutritional status also determine the amount of a nutrient that the body may use, store or excrete, the term “bioavailability” is usually referred to as the fraction of a given compound or its metabolites that
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reaches the systemic circulation without considering bioactivity (Holst & Williamson, 2008; CarbonellCapella et al, 2014).
In vitro models based on human physiology have been developed as simple and reproducible tools to study digestive processes (i.e. stability, micellization, intestinal transport) and to predict the bioavailability of different food components (i.e. carotenoids). Overall, reasonable correlations between in vitro bioaccessibility, in vivo observations and results from human bioavailability trials have been reported. Broadly, for lutein, β-carotene, lycopene and β-cryptoxanthin both qualitative and (semi)quantitative correlations have been found suggesting that in vitro bioaccessibility can be indicative of the amount available for uptake in the (in vivo) gastrointestinal tract (Reboul et al, 2006; GranadoLorencio et al, 2007a, 2007b; Maiani et al, 2009; Bohn et al, 2015). Caco-2 cells have been also used as surrogate for enterocytes and human absorption, including chylomicron secretion (Failla & Chitchumroonchokchai, 2005). Nevertheless, different Caco-2 cell lines may generate distinct metabolite
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ACCEPTED MANUSCRIPT profiles and these cells take up a wide range of carotenoids, even those not detected in human serum (i.e. neoxanthin, xanthophyll esters) (Sugawara et al. 2001; During et al, 2002; Failla & Chitchumroonchokchai, 2005), a fact that compromise its comparability with in vivo (human) situations. In general, the behaviour of carotenoids under in vitro gastrointestinal conditions does not fully explain the changes observed in vivo (Bohn et al, 2015). In vitro models may provide relevant information regarding food factors influencing bioavailability but they cannot totally simulate in vivo human situations, especially those concerning biological variability, timing of intervention, postprandial metabolism and distribution, and biological effects upon regular intake (Granado-Lorencio et al, 2011). Overall, carotenoid bioavailability in animals and humans have been approached using different methods
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including balance studies, dose-effect relationships and isotope-labeled compounds plus compartment modeling (Ball, 1998; Failla & Chitchumroonchokchai, 2005; Bohn, 2008). However, regardless of the method used, absorption, status, distribution and storage in humans are assessed with the use of
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biochemical markers.
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Within this context, the present article aims to review several methodological, host-related and modulating factors relevant on assessing and interpreting carotenoid bioavailability. We briefly describe approaches and variables affecting the assessment of the bioavailability of carotenoids in humans,
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including the type of markers, analytical issues, the time of exposure, qualitative and quantitative issues of the response as well as modulating and confounding factors affecting the interpretation of data. Overall, the paper provides an overview from a nutritional biochemistry perspective regarding the use of
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markers to assess carotenoid bioavailability. 2.- BIOMARKERS
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A key issue in the design of human nutritional studies is the choice of the right markers which, in turn, depends on the objective of the study. Overall, for essential nutrients, nutritional status (as a
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surrogate of bioavailability) can be approached by clinical, dietetic, anthropometric and biochemical markers. Nevertheless, for carotenoids, and especially for non-provitamin A carotenoids, most of these approaches are not applicable since nutritional status cannot be properly defined and inadequate intake
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does not result in biochemical or clinical signs of deficiency. As for other micronutrients and phytochemicals, biomarkers for carotenoid bioavailability can be broadly divided into direct, biochemical or “analytical” markers and indirect, physiological or “functional”
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indicators. Usually, analytical markers refer to biochemical indicators of intake and/or status (exposure) of a given nutrient or food component. In turn, functional markers evaluate the effect or a biological activity associated with a nutrient or its absence. 2.1.- Biomarkers of exposure Traditionally, the assessment of carotenoid exposure in humans has been performed by dietary or biochemical methods, both of which have advantages and limitations. In general terms, markers of exposure evaluate biological accessibility although a critical issue is to assess to what extent the marker reflects actual dietary exposure [Ellwood et al, 2014]. Overall, concentrations of carotenoids in serum and tissues are considered more reliable than dietary methods since they provide a more accurate measurement of the amounts available to tissues (Van den Berg et al, 2000; Maiani et al, 2009; Raiten et al, 2011; Scalbert et al, 2014; Tanumihardjo et al, 2016). However, serum carotenoids may be influenced by the bioavailability and in vivo metabolic changes and thus, they do not represent a true
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ACCEPTED MANUSCRIPT measurement of dietary exposure. Even so, blood concentrations of biomarkers provide direct relationships between nutrient input, status and availability for tissue metabolic requirements (Thurnham & Northrop-Clewes, 2016). Tissues also reflect exposure to carotenoids. Adipose tissue is thought to be a more stable depot of carotenoids although this requires invasive approaches (biopsies). Similarly, carotenoids accumulate in human skin and supplementation leads to increases in carotenoids content. Thus, skin carotenoid status (measured by reflectance methods or Raman spectroscopy) may be used as an objective biomarker of dietary intake (bioavailability), although diet explains only some of the variation in this biomarker (Mainke et al, 2010; Mayne et al, 2013). Also, non-invasive tests (i.e. heteroflicker
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photometry) have been used to measure macular pigment optical density (MPOD) which provides information on long-term lutein (and zeaxanthin) exposure (Beatty et al. 2001; Bernstein et al, 2002). other (non-ocular) tissues is uncertain (Granado et al, 2003).
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2.2.- Functional biomarkers
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However, because of the selective deposition of these carotenoids in this tissue (retina), its relevance to
Functional markers are considered to be indicative of biochemical/physiological changes as a
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result of the exposure. Overall, functional markers may be related with long-term (i.e. cumulative) exposure but they are subject to confounding factors and may be influenced by other micronutrients (i.e. methyl group donors and B vitamins for DNA methylation, interactions of divalent cations during
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absorption, copper/zinc-dependent enzyme activity) (Elmafda & Meyer, 2014). Noteworthy, many bioavailability studies with carotenoids are performed using “natural” sources (i.e. extracts, whole and fortified foods) rather than single compounds (i.e. supplements) so that other dietary components are co-ingested (collinearity) making difficult the interpretation of results in terms of comparability and
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“selectivity” (as a criteria of causality).
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Examples of functional tests used to evaluate carotenoid bioavailabity /response are shown in table 1. Under normal circumstances, the functional measures correspond to bioavailability, but the relationship may be influenced by host factors (Degeraud et al, 2015). Functional bioefficacy can be also measured by determining the rate by which deficiency symptoms are cured or by using other measures
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specific to the nutrient's function. As some carotenoid are converted into retinol, functional improvement in vitamin A status could be used as a surrogate of the intervention with carotenoids by assessing visual function or ocular indicators (Failla & Chinchorookchoktai, 2005; Degeraud et al, 2015). In addition,
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bioefficacy can be assessed using blood retinol concentration although serum retinol is homeostatically controlled by the liver (Tanumihardjo et al, 2016). In general, functional tests show several constraints such as the lack of standardization, the potential synergism between nutrients, the absence of accepted reference ranges or the influence of polymorphisms unrelated to the bioavailability of the nutrient (i.e. enzymes). Additionally, although these methods may provide an index of inadequacy (i.e. dark adaptation), they may not be responsive within the full range of normal intake or above [Raiten et al, 2011; Tanumihardjo, 2004]. Importantly, changes in markers of exposure or status (i.e. increase in serum levels) may not be necessarily followed by changes in markers of function or effect (i.e. reduction of markers of oxidative stress) and thus, both types of markers are not comparable or interchangeable since they provide different information regarding the bioavailability of a given compound (i.e. availability or associated biological action).
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ACCEPTED MANUSCRIPT 3.- METHODOLOGICAL ISSUES 3.1.
Analytical points
Naturally occurring dietary carotenoids include a wide range of chemical forms such as carotenes, free xanthophylls as well as mono- and di-esters of several xanthophylls mainly found in fruits (i.e. citrus fruits, papaya, goji berries) (Van den Berg et al, 2000; Maiani et al, 2009; Bohn et al, 2015). Although it is essential to identify the carotenoid profile in foods as it is what we may find in vivo (i.e. plasma, tissues), carotenoids may be metabolized in the body. In addition, during food processing, loss of carotenoids may occur and levels of cis-isomers may increase due to the isomerization of the trans-forms. However, many bioavailability studies are based on the measurement of intact (i.e. total
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and free forms) carotenoids in the blood or tissues and hence, so that characterization and quantification of carotenoid changes (i.e. cis-isomers, in vivo metabolites) is necessary to assess accurately
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bioavailability (Arathi et al, 2015) and the analytical approach becomes essential.
Overall, although the best approach may depend on the matrix and the objective of the study,
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analyses of carotenoids from biological matrices involves several steps including sample collection/ preparation, extraction (with or without saponification), separation, detection and quantification. However, despite new developments and improvements in separation techniques (i.e. columns, mass
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spectrometry detectors), analytical inaccuracies and artifacts (i.e. sample stability, extraction procedures, lack of standards) may be still present (van den Berg et al, 2000; Arathi et al, 2015; Bohn et al, 2015). In general, for food analyses and in vitro digestion systems, extraction with and without
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saponification may be used so that information regarding free forms and total content of xanthophylls may be obtained (i.e. to assess the degree of hydrolysis in vitro). A saponification step, however, is not usually performed with serum or triacylglycerol-rich lipoprotein fractions (TRL) due to the (expected)
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absence or very low presence of xanthophyll esters and the potential losses during the procedure. In clinical practice, carotenoids analysis (mostly β-carotene) is not routinely but exceptionally
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performed (i.e. genetically-based incapacity to convert β-carotene into retinol) and the best practice includes the use of fasting serum collected in plain or gel separator or anti-coagulated tubes (heparin, EDTA) (Greaves et al, 2014). In this sense, using serum and Li-heparin plasma, an acceptable degree of agreement has been reported for most serum carotenoids except possibly for minor components (i.e.
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zeaxanthin, α-cryptoxanthin) especially at the lower end of the concentrations distribution (OlmedillaAlonso et al, 2005). Thus, the type of specimen (i.e. serum, Li-heparin plasma) does not appear to be a matrices).
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relevant analytical constraint, at least in relative terms or for comparative purposes (i.e. different food
As a laboratory medicine best practice, C18 columns and UV/Vis detection are recommended for the analysis of vitamin A and β-carotene, although mass spectrometry is recommended as an alternative detection method (Greaves et al, 2014). Traditionally, reversed-phase HPLC on C18 columns have been widely used for separating carotenoids while polymeric C18 phases have provided an acceptable selectivity for similar carotenoids such as the cis- and trans- forms and structural isomers (i.e. lutein and zeaxanthin), specially under gradient elution conditions (Granado et al, 1991; Olmedilla et al, 1997). Nevertheless, non-endcapped columns with triacontyl (C30) ligands were developed specifically for a better isomer resolution (Sander e tal, 1994) and have become widely used for research purposes (Arathi et al, 2015; Gupta et al, 2015; Bohn et al, 2015). Morever, despite it usually requires longer run times, limiting its utility in large studies and clinical practice, rapid and sensitive methods have been also developed (Gupta et al, 2015). Alternatively, the development of columns with particles < 2 µm has provided new approaches to resolve carotenoid mixtures in much shorter times (ultra-fast HPLC),
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ACCEPTED MANUSCRIPT improving resolution, increasing sensitivity and reducing substantially the associated costs (Granado et al, 2010a). In addition, the occurrence of carotenoids cleavage products is crucial to assess their potential bioactivity. In this context, carotenoids in biological samples are frequently detected below nanomolar concentration and metabolites exist at picomolar concentration (Khachik et al,1997). Therefore, improvement in analytical techniques to resolve and identify retinyl esters and carotenoid metabolites under in vivo conditions (i.e. TRL) requires sensitive methods (i.e. HPLC coupled with MS/MS or
NMR) as well as the availability of appropriate standards (Kopec et al, 2013; Arathi et al, 2015; Gupta
3.2.
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et al, 2015; Bohn et al, 2015). Timing of intervention.
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In general, single dose studies aim to assess bioavailability, pharmacokinetic and acute effects while medium/long-term trials are intended to evaluate chronic (cumulative) exposure, functional changes or
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modifications of risk factors. The information obtained is somewhat different since it is interpreted in terms of postprandial response/metabolism (<48-72h), and repletion and/or saturation processes (> 1
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week), respectively (Granado-Lorencio & Olmedilla-Alonso, 2003).
Overall, a relationship exists among the type of marker (analytical or functional), the biological specimen needed and the time required for a change (FIGURE 1). Broadly, recent exposure may be
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assessed by analytical markers measured in biological matrices with relatively “fast turnover” (i.e. postprandial TRL fraction, serum, urine, feces, breath, breast milk, bile). On the contrary, for cumulative exposure (i.e. multiple dose studies), analytical markers measured in samples with “longer turnover” (i.e. lymphocytes, buccal mucosa cells, colonocytes) or storage tissues (i.e. adipose tissue)
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are preferable. Similarly, for functional tests, the markers will depend on the intervention time so that
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this should be long enough to provoke a change in the markers and matrix selected. Multiple-dose intervention studies may be useful for comparing population groups, food sources, ingredients or functional effects but this approach cannot be used to quantify the rate and extent of carotenoid absorption (Faulks & Southon, 2005). Moreover, as the time of intervention increases,
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changes in biomarkers may reflect better a cumulative effect (i.e. predictive value for a clinical outcome) although the potential confounding factors (i.e. tissue metabolism), the rate of drop-outs, and the costs increase as well. Additionally, biomarkers with a high methodological variability (i.e. subject-dependent
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carotenoid metabolites) often require a higher number of participants or repeated measures to account for within-person variations and to provide a more reliable index of exposure, status and response [Ellwood et al, 2014; Rhagavan et al, 2016]. 3.2.1.
Postprandial response, clearance and excretion
The preferred method of measuring carotenoid absorption and bioconversion is to isolate the triacylglycerol-rich lipoprotein plasma fraction (TRL) which contains newly absorbed carotenoids and formed retinoids (Van den Berg et al, 2000; Failla & Chitchumroonchokchai, 2005; Carbonell-Capella et al, 2014). However, it is necessary to discriminate between newly absorbed from endogenous carotenoids and to consider the clearance kinetics, since these factors may affect the estimated area under the curve (AUC) (Faulks & Southon, 2005). In this context, it is important to note that the use of large-doses and medium-/long-term supplementations may lead to non-physiological effects by altering the normal bioavailability, metabolism and clearance rates, or lead to non-linear responses (Welch et al,
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ACCEPTED MANUSCRIPT 2011). In addition, carotenoids (especially xanthophylls) may be transferred between lipoproteins and be taken up by extrahepatic tissues before reaching the liver and thus, analyses of this fraction may not provide an accurate estimate of the “true” relative absorption (van den Berg et al, 2000; Furr & Clarke, 2003). Plasma response (kinetics) of individual carotenoids also differs so that the timing of sample collection becomes essential. For example, in humans, the time for peak concentrations in TRL fractions was shorter for lutein (about 2h) than for β-carotene and lycopene (about 4-6 h) (O´Neill & Thurnham, 1998) while astaxanthin showed two peaks (at 7 and 16h) coinciding with peak plasma triglyceride concentrations (Furr & Clarke, 2003). Normally, after oral dose, peak concentrations of carotenoids in
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plasma occur at 6-48h (depending upon the dose and the frequency of measurements) which is slower than the time of appearance of cholesterol and triglycerides after a meal (usually 4-6h). Moreover, there is a second peak in plasma carotenoid which seems to accompany the later meal, making difficult to
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measure accurately the AUC (van den Berg et al, 2000; Faulks & Southon, 2005).
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Carotenoids also display distinct half-life in humans which may be related to differential deposition in target tissues, utilization or metabolism. It has been reported that all-trans-lycopene displays a shorter half-life than the cis- isomers (Moran et al, 2015). Similarly, compared to lycopene,
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the clearance rate of phytoene from chylomicrons was slower (Moran et al, 2016) while that of capsanthin appeared to be much faster (Oshima et al, 1997). Additionally, in the absence of carotenoid intake, plasma levels fall to 10-75% of baseline concentrations after 2-3 weeks, with a faster decline
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during the first 2-7 days (Rock et al, 1992). Importantly, many bioavailability studies include a “run-in” period (usually, a low/free carotenoid diet) to accommodate volunteers and ensure compliance. However, this “depletion” diet may lead to overestimate the “true” magnitude of the response by
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considering baseline concentrations as those after a clearance period. The routes of elimination may be also relevant when assessing carotenoid bioavailability.
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Several studies indicate that more than 70 % of the carotenoids may remain in the final digesta (Maiani et al, 2009; Bohn et al, 2015) and consistently, kinetic studies in man showed that the cumulative elimination of 14C in faeces was 51% of the administered 14C-β-carotene dose (Ho et al, 2007). In this sense, the presence in human faeces of free /esters forms and cis- /trans- isomer ratio reflecting the
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diet supports the use of faecal carotenoid profile as a suitable biomarker (and non-invasive approach) regarding responsiveness (i.e. compliance), nutrient stability and availability of chemical forms reaching the colon (Hernandez-Alvarez et al, 2015). However, although it is easy to collect fresh fecal samples,
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interpretation should be made cautiously since fecal carotenoids reflect different sources including unabsorbed dietary content, sloughed gastrointestinal cells, carotenoids excreted in bile and, possibly, microbiota-related metabolites. Finally, carotenoid excretion via the urine has long been considered to be very low. However, tracer studies have shown that 16–30% of orally administered 14C-β-carotene was excreted in urine within 72hours-21 days (Ho et al, 2007). Similarly, after a single microdose of 14C-lycopene, radioactivity was detected in exhaled carbon dioxide (3%) and urine (18%) within 4 h and 12 h, respectively (Ross et al, 2011). Thus, it is likely that urinary compounds represented oxidized and conjugated metabolites (Failla & Chitchumroonchokchai, 2005). Additionally, carotenoids are eliminated without modifications into bile (Leo et al, 1995), although the occurrence of a potential enterohepatic recirculation is unknown. Similarly, carotenoids in breast milk reflects serum profile (Khachick et al, 1997; Canfield et al, 2003) and constitutes another route of elimination.
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ACCEPTED MANUSCRIPT 3.3.
Biological Matrix.
Both serum and tissue carotenoid concentrations are used as biomarkers of recent and longterm exposure, respectively. Humans are yellow-fat animals and accumulate carotenoids reasonably well (i.e. adipose, liver and skin). For example, carotenoid intake correlates with adipose tissue concentrations of α-carotene, β-carotene, β-cryptoxanthin, cis-lycopene, and total carotenoids (Chung et al, 2009) while dietary carotenoids such as α-, γ-, β-carotene, lycopene, lutein, zeaxanthin and their isomers may be found in human skin (Wingerath et al, 1998). However, selective deposition of some carotenoids in certain tissues is well-known. Lycopene is
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concentrated in the prostate, testes or liver (Moran et al, 2013) while lutein and zeaxanthin are especially abundant in retina (macula) where they represent 80-90% of the total carotenoids (Handelman et al, 1992). The brain, like the retina, also appears to accumulate xanthophylls and
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correlate with serum carotenoids so that these have been proposed as biomarkers to predict brain carotenoid concentrations (Johnson et al 2011; Hammond, 2015). Similarly, concentrations of
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carotenoids in breast milk may be used as an index of maternal dietary intake (Canfield et al, 2003; Lipkie et al, 2015; Tanumihardjo et al, 2016) although they are associated with fat content, vary with the lactation stage and lutein and zeaxanthin levels are higher than those of β-carotene, in contrast to
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their concentrations in maternal plasma (Furr & Clarke 2003; Canfield et al, 2003). Importantly, when planning bioavailability studies in humans, ethical considerations are mandatory so that invasiveness for sample collection becomes essential. While adipose tissue and liver
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may be adequate matrices to assess long-term intake/exposition, other (non-invasive) tissue samples may be easily accessible (i.e. buccal mucosal cells, exfoliated colon cells) despite factors such as cell turnover must be considered. Alternatively, macular pigment optical density (measured by heterochromatic flicker photometry) and skin carotenoid status (assessed by reflectance based methods
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and Raman spectroscopy) may be used as markers of long-term dietary exposure. 4.- MARKERS OF CAROTENOID BIOAVAILABILITY 4.1.- Qualitative features
The influence of the diet on the profile and levels of carotenoids in serum has been known for
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decades and it is widely accepted that serum carotenoids represent good biomarkers of fruit and vegetable intake at ecological level (Olmedilla et al, 2001; Al-Delaimy et al, 2005; Maiani et al, 2009,
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Bohn et al, 2015).
Humans are considered indiscriminate carotenoid absorbers (Goodwin 1984), meaning that we are capable of absorbing a wide range of carotenes and xanthophylls. Approximately, 40–50 carotenoids are available in commonly consumed fruits and vegetables which can be divided into carotenoid epoxides, mono- and di-hydroxy-carotenoids, hydrocarbon carotenoids and carotenol acyl esters. From these, a significant proportion can be absorbed through the intestine, incorporated (unaltered) into chylomicrons and be available systemically. Broadly, bioavailability partly depends on the carotenoid structure (i.e. provitamin A capacity) and, in general, polar carotenoids are preferably incorporated into mixed micelles and tend to be of higher bioavailability, as may be the case for lutein vs β-carotene, free vs. esterified xanthophylls and cis- vs trans-forms (Bohn, 2008). However, up to∼34 carotenoids (including geometric isomers, metabolites and acyclic colorless carotenes) have been identified in human serum and tissues (Khachick et al, 1997) (TABLE 2). Nevertheless, just 6 of these, α-carotene, β-carotene, lutein,
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ACCEPTED MANUSCRIPT zeaxanthin, β-cryptoxanthin and lycopene comprise ∼90% of that total number (Bieri et al, 1985; Olmedilla et al, 2001). Epoxy-carotenoids Epoxy-carotenoids (i.e. neoxanthin, violaxanthin) are ingested together with lutein from green vegetables but it has been long considered not to be absorbed (possibly destroyed upon gastrointestinal conditions or rapid metabolism) due to their absence in serum and tissues (Khachick et al, 1991). Nevertheless, under in vitro conditions, free epoxy-carotenoids have been found in supernatants (Granado-Lorencio et al. 2007a) and Caco-2 cells were reported to take up neoxanthin and fucoxanthin
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(Sugawara et al. 2001).
In humans, 5, 6-epoxy-β-carotene may be absorbed as indicated by their presence in circulating
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blood (Barua et al, 1999) while epoxy-xanthophylls such as lutein 5,6-epoxide, violaxanthin and capsanthin-5, 6-epoxide, or any of their metabolites, were not detected after a single-dose (Barua and
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Olson 2001; Pérez-Gálvez et al, 2003; Kotake-Nara et al, 2011). Similarly, even after 1-week intake of epoxy-xanthophyll-rich diets, plasma concentrations of neoxanthin, its metabolites (neochrome stereoisomers) and fucoxanthinol (a gastrointestinal metabolite of fucoxanthin) remained very low low (Asai et a, 2004; Kotake-Nara et al, 2011).
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(about 1 nmol/l) or absent, suggesting that the plasma response to dietary epoxy-xanthophylls was very
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Keto, keto-hydroxy- and carboxylic carotenoids
Upon acute ingestion, keto and keto-hydroxy-carotenoids (i.e. astaxanthin, capsanthin, capsorubin, sapotexanthin, cryptocapsin) have been found in human chylomicrons and plasma (Oshima
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et al. 1997; Odeberg et al. 2003; Coral-Hinostroza et al. 2004; Chacón-Ordoñez et al, 2017). Astaxanthin and several radio-labeled metabolites were reported in humans reaching peak levels at 11.5
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h after administration (Kistler et al, 2002) while capsanthin and capsorubin (i.e. paprika oleoresin) appear to be less bioavailable compared to common carotenoids also present in the resin (Oshima et al, 1997; Etoh et al, 2000; Perez-Galvez et al, 2003). Also, upon supplementation, naturally occurring dicarboxylic apo-carotenoids i.e. bixin and its metabolite (norbixin), have been found in human blood,
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showing maximal concentrations at 2-4h (bixin) and 4h (norbixin) and a complete clearance by 8h and 24h (for bixin and norbixin, respectively) (Levy et al, 1997).
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Colourless carotenes and precursors Postprandial absorption of phytoene, phytofluene, ζ-carotene and neurosporene (i.e. chylomicrons) in humans appears to follow the same absorption time-course as lycopene and other carotenoids (Cooperstone et al, 2015). Additionally, both phytoene and phytofluene have been identified in breast milk and human tissues and appeared to be more bioavailable and more efficiently deposited in tissues compared to other carotenoids (i.e. lycopene) (Melendez-Martinez et al, 2015; Moran et al., 2016). Xanthophyll esters Foods containing xanthophyll esters (i.e. fruits) may transfer free xanthophylls more efficiently than those containing only free forms, a fact and that might explain partly the in vivo provitamin A value of β-cryptoxanthin-containing foods (Burri, 2015; Burri et al, 2016). However, the available data are inconsistent and suggest that the degree of bioavailability of free and ester forms of carotenoids may
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ACCEPTED MANUSCRIPT depend on the carotenoid and the matrix assessed. Thus, comparative studies showed an enhanced bioavailability for zeaxanthin dipalmitate compared to the non-esterified form (Breithaupt et al. 2004) while comparable bioavailability were observed for free and β-cryptoxanthin esters (Breithaupt et al, 2003). For lutein, a higher bioavailability of ester forms has been reported (Bowen et al, 2002) although no differences has been also found (Chung et al, 2004) and lutein response from supplements containing free lutein was greater than that from those containing lutein esters (Norkus et al, 2010). Under in vitro conditions, ester forms have been found in the aqueous-micellar phase and minor, although quantifiable, amounts have been reported to be taken up by Caco-2 cells (GranadoLorencio et al. 2007a; Failla & Chitchumroonchokchai, 2005). Thus, the postprandial absence of
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xanthophyll esters (i.e. lutein, β-cryptoxanthin and astaxanthin) in humans has led to the assumption that xanthophyll hydrolysis is a prerequisite for xanthophyll absorption (Wingerath et al, 1995; Breithauptet al. 2004; Schlattereret al. 2006; Granadoet al. 2010; Burri, 2016). Nevertheless, lack of
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sensitivity to detect low amounts of esters in TRL fractions and/or first-pass metabolism during absorption (i.e. hydrolysis at the enterocyte) may also explain their postprandial absence. Only upon
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long-term supplementation, the presence of xanthophyll esters in serum have been reported (Granado et al, 1998) although esters of lutein, zeaxanthin, anhydrolutein and α- and β-cryptoxanthin are
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normally present in human skin (Wingerath et al, 1998). Cis-isomers
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Most carotenoids in plants exist in the all-trans configuration although food processing (i.e. heating, canning) can induce cis-isomerization. However, the cis-trans ratio of carotenoids in chylomicrons and serum remains fairly constant, regardless of their proportion in the diet, the population studied or the isomer distribution in the supplements provided (Stahl et al. 1995; You et al.1996; Olmedilla et al.
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2002; Granado-Lorencio et al, 2007b).
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Both the cis-isomer profile and the cis/trans ratio vary in tissues as well. Apparently, isomerization of β-carotene did not occur in the gastrointestinal tract and both all-trans and 9-cis β-carotene are similarly absorbed (Faulks et al, 1997). However, after an oral dose containing all-trans and 9-cis-βcarotene, humans show a much higher level of all-trans-β-carotene than 9-cis-isomer in chylomicrons
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and very-low density lipoproteins (VLDL) (Stahl et al, 1995). In fasting conditions, 13-and 15-cis-βcarotene are found in serum at only 5-10% of the all-trans isomer while a higher proportion if cisisomers are found in tissues (10-40%) (Stahl et al, 1992; Van Vliet, 1996; Faulks & Southon, 2005). In
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addition, 9-cis-β-carotene has been reported in tissue samples but not in serum (Stahl et al, 1992). Isomeric composition of lycopene also differs in the diet, serum and human tissues. It is well known that all-trans-lycopene predominates in the main dietary sources of lycopene (i.e. tomatoes and tomato products), but plasma and tissues contain relatively greater concentrations of cis isomers. All-translycopene accounts for 95% in foods, about 65% in chylomicrons and 45-50% in serum with at least three cis-isomers (9-, 13-, and 15-cis) being present in serum. Although this pattern may suggest that cis-lycopene isomers may be more bioavailable (Bohn, 2008), studies in humans with C13-lycopene have revealed that post-absorptive trans-to-cis-lycopene isomerization, and not the differential bioavailability of isomers, drives tissue and plasma enrichment of cis-lycopene (Ross et al, 2011; Moran et al, 2015). On the contrary, it has been reported that astaxanthin cis-isomers were selectively absorbed into plasma, comprising ~32% of total astaxanthin postprandially (Coral-Hinostroza et al, 2004).
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ACCEPTED MANUSCRIPT Overall, although several mechanisms may promote a selective enrichment of cis-isomers of some carotenes and xanthophylls (i.e. cis-isomers are less likely to crystallize, preferentially micellarized or more readily taken up by intestinal cells) (Cooperstone et al, 2015), these apparently affect differently to each carotenoid (i.e. the predominance of all-trans-β-carotene vs cis-lycopene in serum). 4.1.1. In vivo metabolites as surrogates of carotenoid bioavailability Approximately, ~ 10% of dietary carotenoids can be converted into vitamin A and thus, postprandially, provitamin and non-provitamin A carotenoids as well as retinyl esters are incorporated into chylomicrons. During postprandial states, the measurement of retinyl esters (mostly retinyl
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palmitate and esters with other long-chain fatty acids) and carotenoids in TRL fractions has been frequently used to assess both the degree of absorption and conversion (AUC), at least in comparative terms. Under fasting conditions, however, retinol (bound to retinol binding protein and transthyretin) is
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the major circulating form and retinyl esters represent<10% of the total vitamin A in serum (Tanumiharjdo et al, 2016). Additionally, serum retinol levels are homeostatically regulated so that its
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use as a marker of bioefficacy is limited.
Dehydration products (i.e. anhydrolutein) have been reported in human serum and tissues and
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suggested to be non-enzymatically formed in the stomach under acidic conditions (Khachick et al, 1991, 1995, 1997). Other metabolites of lutein and zeaxanthin (i.e. 3′-Epilutein, meso-zeaxanthin, 3´oxolutein, 3 -methoxy-zeaxanthin) have also been reported in human serum, milk and ocular tissues
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(Khachik et al., 1995, 1997; Bernstein et al., 2001; Bhosale et al, 2007; Bhosale & Bernstein, 2005;Thürmann et al, 2005). Additionally, oxidative metabolites of lutein characterized as mono-keto, mono-hydroxy and di-keto-carotenoids have been also reported in human serum (Khachick y cols, 1991; 1995; 1997). Moreover, since these keto-carotenoids are not widely found in foods and increase in
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serum upon lutein supplementation, it is suggested that these carotenoids are formed in vivo (Khachik et al, 1997; Granado et al, 1998). Similarly, the presence and in vivo formation of lycopene epoxides
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(i.e. 2, 6-cyclolycopene-1,5-diols) in human tissues such as prostate, lung and colon have been also reported (Khachick et al, 1997; 2002). Thus, the occurrence of these metabolites in serum and tissues indicates that several carotenoids may undergo chemical modifications under in vivo conditions. Apo-carotenoids (i.e. β-apo-carotenoids and apo-lycopenoids) are present in the diet (i.e.
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processed mango juice, dried apricots, tomato extracts and paste) and can be formed via autoxidation, thermal degradation or during food processing (Eroglu et al, 2013). Nevertheless, it has been reported the presence of β-apo-carotenals and β-apo-carotenone in human and animal blood and tissues (Wang
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et al, 2015) and their formation under in vitro conditions as a result of lycopene and xanthophylls metabolism (Goupy et al 2012; Mein et al, 2011; Sharoni et al, 2012). In this context, excentric cleavage of ingested β-carotene occurs in vivo and the formation of several apo-carotenoids have been tentatively identified in humans (i.e.β-apo-8´-carotenal, β-apo-13-carotenone) (Ho et al, 2007; Eroglu et al, 2012). Moreover, the appearance of 14C- β-apo-8´-carotenal in plasma 3 days after dosing suggests that β-apo-carotenals are formed in peripheral tissues (Ho et al, 2007). Similarly, lycopene metabolites (lycopenoids) have been reported in serum and human milk (Lindshield et al. 2007) and several apo-lycopenals (i.e. 6´-, 8´-, 10´-, 12´- and 14´-) were found in human plasma of subjects after consuming tomato juice (also containing lycopenals), although whether the plasma apo-lycopenals originated from enzymatic cleavage of lycopene or from consumption of apo-lycopenals containing fruits and vegetables is not clear (Kopec et al.2010; Eroglu et al, 2013). Overall, despite different carotenoid metabolites (i.e. keto-carotenoids, cyclo-lycopene diols, apo-carotenoids) have been found in serum and tissues and may respond (increase) upon
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ACCEPTED MANUSCRIPT supplementation, they have not been frequently assessed so that their utility as biomarkers of bioavailability (exposure or effect) remains to be established. 4.2.- Quantitative considerations Many designs to assess carotenoid bioavailability yield information regarding relative bioavailability (compared to a reference dose or control) and thus, studies using different methodological approaches (i.e. food matrix, dose, timing) can lead to different results when determining fractional absorption (van den Berg et al, 2000; Granado-Lorencio & Olmedilla-Alonso, 2003; Failla &
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Chitchumroonchokchai, 2005). For carotenoids, both absorption of unaltered compounds (provitamin and non-provitamin A carotenoids) and conversion into retinol (first-pass metabolism) must be considered. Human studies
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performed in the 1960´s identified retinyl esters as the major metabolites of β-carotene in the lymphatic system (61-80% of labeled compounds), with variable amounts of unaltered β-carotene (up to 30%)
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(Goodman et al, 1966; Blomstrad et al, 1967). Using postprandial (TRL) models and a 1:2 molar conversion factor (β-carotene/retinol) percentages have been established between 35-71% (Van Vliet et al, 1996; Granado et al, 2001) while using labeled β-carotene and 1:1 molar ratio, about 20% of the
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absorbed β-carotene was converted into retinol (Novotny et al, 1995). More recently, the estimated βcarotene bioefficacy (a combination of both absorption and conversion) was estimated at 13.5%
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±6.02%, although a high inter-individual variation (CV: 44%) was also observed (Green et al, 2016). In contrast to serum retinol which is tightly (homeostatically) controlled, serum concentrations of carotenoids in humans are not regulated. Overall, the absorption efficacy of carotenoids is highly variable ranging between 5-80% (Parker, 1996; Erdman et al, 1993; De Pee & West, 1996). Assuming a
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dose-response effect, a proportional variation of serum /tissue levels of carotenoids with increasing amounts of intake is expected (i.e. “linearity”), at least at dietary achievable levels. In this sense,
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significant but highly variable correlations between dietary intake and serum concentrations have been reported (r= 0.1- 0.7) depending on the carotenoid and the study (Furr & Clark, 2003; Al-Delaimy et al, 2005). Consistently, carotenoid profile and levels in tissues (i.e. adipose, prostate, liver, colon cells) seems to respond to nutritional interventions although the magnitude of the associations differ for
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various tissues (Maiani et al, 2009; Sen et al, 2013; Bohn et al, 2015). Factors affecting the magnitude of the correlations are both methodological (i.e. dietary
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assessment methods, food carotenoid databases) and physiological (i.e. first-pass metabolism, tissue deposition). Alternatively, due to the lack of robust linear relationships, (semi)quantitative relationships have been approached by classifying subjects according to their level of exposure (i.e. quartiles of intake and serum levels) so that an acceptable degree of agreement (correct classification) may be obtained. Even so, the concordance between intake level and serum concentrations should be interpreted cautiously since they do not necessarily reflect the relative amount of structural and geometrical isomers. For example, levels of lycopene in prostate correlated with post-intervention plasma lycopene concentrations (r 0·60, P =0·001) but plasma and prostate cis-isomers accounted for 47 and 80 %, respectively (Grainger et al, 2015). Similarly, in serum, the lutein: zeaxanthin ratio is about 3:1 whereas in macula, this ratio reaches values of up to 1:2. Nevertheless, concentrations of lutein and zeaxanthin in serum and tissues (i.e., macula) increase on consuming lutein-rich foods or supplements. Moreover, this increment parallels the increase in MPOD (macular pigment optical density) and the improvement in visual function (Trieschman et al, 2007; Schalch et al, 2007; Olmedilla et al, 2003), demonstrating
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ACCEPTED MANUSCRIPT consistently a relationship between serum and tissue markers (lutein levels, MPOD) and functional indicators (i.e. visual function) (Olmedilla et al, 2003; 2014). Compared to drugs, however, the (functional) effect derived from nutritional interventions is expected to be small although markers of exposure (i.e. serum carotenoids) may change significantly. Due to the endogenous presence of carotenoids, large doses have been frequently used to provoke a measurable response. However, this approach has been criticized as they may alter the physiological response and assume non-saturable models and linear dose-responses. Additionally, effects upon intervention may be related to nutritional status, the dose and the time of the intervention, all of which may influence the response and even lead to a “ceiling effect” (Olmedilla et al. 2002; Schlatterer et al.
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2006). Moreover, postprandial behaviour does not predict long-term effects, and acute (single dose) responses usually show larger inter-individual variability than chronic (multiple) intakes (Borel et al. 1998; Olmedilla et al. 2002; Granado-Lorencio & Olmedilla-Alonso 2003; Faulks & Southon, 2005). For
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example, using a beverage enriched in lutein at two levels, a greater postprandial AUC was obtained with the high dose although the percentage of absorption was not different when standardized to the
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amount supplied. Nevertheless, upon regular consumption, serum increments at 7 and 14 days were statistically higher on consuming the high-dose beverage (Granado-Lorencio et al, 2010b). Thus,
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comparison of results from different protocols should be interpreted cautiously. Multiple-dose supplementation studies are also used to assess the bioavailability (i.e. bioactivity or functional effects) of carotenoids in humans so that the linearity of the response becomes an
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important issue. Using lutein, lycopene, α-+ β-carotene, astaxanthin and capsanthin, multiple-dose human studies have reported the presence of a “plateau effect” in serum concentrations of carotenoids (Oshima et al, 1997; Olmedilla et al, 2002; Meinke et al, 2010). Thus, regardless of the underlying mechanisms (i.e. dose, duration of the study, type of biomarker, number of measurements and
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physiological mechanisms), the loss of linearity in the response questions the reliability and utility of serum levels as markers of exposure (bioavailability) above certain concentration threshold. On the
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contrary, consistent with a longer half-life of carotenoids in tissues, carotenoids appear to decrease much more rapid in serum than in tissues (i.e. skin) (Meinke et al, 2010) and thus, levels of carotenoids in tissues could provide, a priori, better linearity and reflect more successfully the cumulative exposure and effects. However, again, the potential presence of carotenoid metabolism in tissues and the cell
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turnover should be also considered.
Overall, in biological systems, large deviations from linearity may occur and, in terms of efficacy
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(function), linear relationships are the exception rather than the rule (Faulks & Southon, 2005). Obviously, when linearity is lacking, the utility of the marker is lost since exposition cannot be reliably related to the effect. Moreover, in many studies, biological samples to measure both analytical and functional markers are usually collected at baseline and at the end of the intervention. Unfortunately, under these conditions, it is not possible to assess when the changes occur, if a minimum amount of the bioactive is needed to get an effect (threshold effect), if a (analytical or functional) change is linear over time, if a saturation (“plateau”) effect is present or for how long the effect is maintained upon discontinuation. 4.3.- Markers of overexposure While clinical signs and biochemical markers of vitamin A toxicity (i.e. increased retinyl esters in serum) exist, this approach is not applicable to carotenoids. Traditionally, dietary carotenoids are considered to be safe and free of secondary effects beyond the potential, benign and reversible
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ACCEPTED MANUSCRIPT yellowish-orange discoloration of the skin (i.e. palms, soles, naso-labial folds) usually referred to as carotenodermia (xanthosis, xanthoderma or pseudoicterus). It is well known that infants are especially prone to develop carotenodermia due to their dietary intake as well as patients taking β-carotene supplements (i.e. erythropoyetic protoporphyria). However, carotenoid-related skin pigmentation may be also observed in subjects with high intake of carotenoidrich foods (i.e. mango, oranges, carrots, tomato products) and supplements. Noteworthy, carotenodermia appears to develop differently according to the carotenoid provided. Upon long-term supplementation in apparently healthy volunteers, carotenodermia was observed in 95% of subjects taking α- + β-carotene, in 40%of those consuming lutein esters and in 25% of the volunteers taking
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lycopene supplements. These findings suggested that carotenoids were not equally deposited in the skin or that other tissues could be primary targets. Importantly, no changes in biochemical or haematological
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indices were associated with this clinical sign (Olmedilla et al, 2002).
In subjects taking tanning pills, it is recognized the potential occurrence of canthaxanthin
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retinopathy, a condition that may lead to long-standing visual changes including significant visual loss (Beaulieu et al, 2013). Upon discontinuation, subjects may be asymptomatic without functional defects but complete disappearance of the golden particles near the macular region could take up to 20 years
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(Hueber et al, 2011). Retinal paramacular cristal deposition and yellow-brown discoloration of the anterior lens capsule have been also reported in subjects with hypercarotenemia (i.e. serum β-carotene 75-325 µg/dl) and carotenodermia (Rahmani et al, 2003), and corneal rings may persist three years
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after the original presentation despite the serum carotene normalizes (Chang et al, 2012). More recently, corneal rings have been reported in four patients receiving vitamin supplementation for agerelated macular degeneration (i.e. AREDS 2 Study) (Eller et al, 2012). These patients had yellow peripheral corneal rings and a subtle yellowing of the skin (carotenodermia). Interestingly, serum
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carotene levels were normal in two of the three cases (<50 µg/dl) in which it was measured (Eller et al,
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2012), suggesting that serum levels may not reflect nor predict reliably the situation in certain tissues. Carotenodermia is usually associated with high levels of carotenoids in blood, a condition referred to as hypercarotenemia (>300 µg carotenoids/dl) (Underwood, 1984). Hypercarotenemia, however, is not a cause of disease but it may be present in some clinical conditions (hypothyroidism,
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diabetes mellitus, renal or liver disease, anorexia nervosa, brain tumor) (Rock & Swenseid, 1993; Olmedilla et al, 1995) and when an incapacity to convert β-carotene into retinol exists (Sharvill, 1970; Lindquist et al, 2007; Maruani et al, 2010). Additionally, the presence of xanthophyll esters in serum has
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been reported in subjects consuming lutein supplements and displaying serum lutein concentrations> 1.05 mmol/l (Granado et al, 1998). Although ester forms represented less than 3% of lutein levels in serum, they were present after one month of supplementation and, interestingly, before the occurrence of carotenodermia.
Overall, however, based on the available evidence, the cutoff point of serum carotenoids above which carotenodermia and corneal rings appear, or the risk increases, cannot be established although it seems to vary for each carotenoid and probably be subject-dependent. 5.
MODULATING FACTORS OF MARKERS OF EXPOSURE AND RESPONSE In general, besides exogenous (food-related) factors, host-related and methodological issues
may influence analytical and functional markers of bioavailability (TABLE 3). Morever, although
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ACCEPTED MANUSCRIPT carotenoids may share chemical structure and properties, these factors may not equally affect the different subclasses of carotenoids. Physiological factors Results from bioavailability studies (i.e. status and response) may be influenced by a variety of host factors including nutritional status (Failla & Chitchumroonchokchai, 2005). In fact, the body can down-regulate bioconversion of provitamin A carotenoids so that high intakes of fruit and vegetables do not usually cause concern for hypervitaminosis A (Tanumihardjo et al, 2016). Also, since carotenoids are transported by lipoproteins and these are affected by several factors, it has been suggested that plasma
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carotenoids status and response should be lipid-standardized (Furr & Clark, 2003). Sex differences in serum levels of provitamin A carotenoids have been reported in several, but not all, populations (Furr & Clark, 2003; Olmedilla et al, 2001) and differential age-dependent responses
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have been observed in human intervention trials (Maiani et al, 2009). However, a decreased chylomicron response of lycopene, but not lutein and β-carotene, was found in older compared to younger subjects
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(Cardinault et al, 2003). Also, body mass index (BMI) and the conversion efficiency of β-carotene into retinol has been correlated so that subjects with more body fat showed a lower capacity of conversion
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(Tang et al, 2010).
Clinical conditions may also affect the bioavailability (status and response) of carotenoids. Hypochlorhydria, achlorhydria and acidic pH diminished the solubility and absorption (AUC) of β-carotene
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while syndromes responsible for fat malabsorption (i.e.cholestasis, pancreatic insufficiency, biliary cirrhosis, cystic fibrosis) are expected to decrease carotenoid bioavailability as well. Other clinical conditions such as type 1 diabetes mellitus has been associated with lower serum retinol but higher
Inflammation
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efficiency (Granado et al, 2001).
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provitamin A carotenoid status, a situation that is not apparently due to an impaired conversion
Nutritional studies are usually performed in control subjects (“apparently healthy”) but the bioavailability of carotenoids (i.e. response) under subclinical conditions (i.e. chronic inflammation) may differ. In general, infection and inflammation may cause an increased demand (intake), and may alter
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metabolism or excretion of nutrients, reducing the availability and transference to tissues (Duncan et al, 2012; Brenashan & Tanumihardjo, 2014). During the acute phase response, more than 200 plasma proteins are modulated of which
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approximately 50% are involved in nutrient transport or regulation of nutrient status (Raiten et al, 2015). Overall, inflammation affects different nutrient biomarkers including retinol and carotenoids. Plasma retinol concentrations may decrease by as much as 50% during infections and systemic inflammation (Duncan et al, 2012;Thurnham & Northrow-Clewes, 2016) while plasma carotenoids may fall rapidly in states with fever and recover when fever declines (i.e. children with pneumonia) (Goodwin, 1984). Additionally, concomitant infection with intestinal helminthes, Helicobacter pylory or other microorganisms may impair carotenoid absorption or utilization (Failla & Chitchumroonchokchai, 2005; Maiani et al, 2009). However, there is not necessarily a linear relationship between nutrient biomarkers and acute response indicators (i. e. C-reactive protein) and thus, on assessing both status and response (i.e. bioavailability), the presence of subclinical chronic inflammation or acute phase response should be considered and, if necessary, adjust the results to provide a reliable measurement (Thurnham & Northrow-Clewes, 2016).
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ACCEPTED MANUSCRIPT Dietary factors and interactions It is well-known that bioavailability of dietary carotenoids is influenced by several dietary factors (De Pee & West, 1996) and a critical step is their liberation from the matrix and its solubilisation into mixed micelles. Natural deposition of carotenoids (i.e. solid-crystalline aggregates, lipid-dissolved forms) in plant and animal-based foods has been shown to be critical on the liberation efficiency (in vitro and in vivo) and thus, on the bioavailability of carotenoids (Schweiggert et al, 2012; 2015). Moreover, ripening-induced changes in the deposition state of carotenoids (i.e. protein-bound in chloroplasts to tubular chromoplasts) may also have a major impact by increasing in vitro bioaccesibility of xanthophylls esters (Hempel et al, 2017).
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It is also known that dietary fat generally increases carotenoid bioavailability and both the amount and the type of fat may affect the bioavailability of some (i.e. lutein), but not all, carotenoids (Roodenburg et al. 2000; Bohn 2008). Very low-fat diets can also decrease carotenoid digestion and
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absorption although β-cryptoxanthin may be less affected than others carotenoids (Burri et al, 2016). Similarly, long chain triglycerides (LCT), compared to medium chain triglycerides (MCT), increase
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chylomicron secretion and may enhance carotenoid absorption. Also, dietary fats/oils rich in unsaturated fatty acids appear to promote carotenoid in vitro bioaccesibility (i.e. β-carotene, lycopene) by enhancing their micellarization during digestion and intestinal transport (Failla et al, 2014), although
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dietary fats rich in saturated fatty acids led to a higher bioavailability of lutein and zeaxanthin, as compared with fats rich in mono- and poly-unsaturated fatty acids (Gleize et al, 2013).
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The presence of absorption modifiers (i.e. metals, fiber), at dietary and supplementation levels, may also affect the bioavailability of carotenoids. Both in vitro and in vivo studies suggest that the degree of xanthophyll esters hydrolysis, the transference of free xanthophylls into micellar phase and dose-adjusted changes of i.e. β-cryptoxanthin may be higher in the presence of iron and milk (Granado-
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Lorencio et al, 2009). Consistently, supplementation with iron, zinc or both have been shown to increase the bioavailability of provitamin A carotenoids from foods, as measured by chylomicron response and
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conversion into retinol (Kana-Sop et al, 2015). On the other hand, plant sterols and water-soluble fibers (i.e. pectin) decrease the absorption of β-carotene, lycopene and lutein (Failla & Chitchumroonchokchai, 2005; Maiani et al, 2009; Bohn et al, 2015). However, although phytosterol intake at the recommended doses (i.e.2 g/day) may lower serum levels of carotenoids (Bañuls et al, 2010), this inhibitory effect may
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be eliminated if co-consumed (Granado-Lorencio et al, 2011). Carotenoid interactions appear to be present between carotenes and xanthophylls and among
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compounds of the same subclasses as they share common mechanisms at different levels (i.e. micellarization, affinity to transporters, provitamin A cleavage, exchange between lipoproteins). Much of the evidence suggests an interaction between ß-carotene and oxy-carotenoids such as canthaxanthin and lutein, and between ß-carotene and lycopene although the magnitude and the direction of the interactions may differ (van den Berg et al, 2000; Reboul et al, 2007; Maiani et al, 2009). Nevertheless, the impact of these interactions will also depend on the relative amounts of carotenoids, the matrix (vehicle) and the protocol used (i.e. single vs multiple dose). Nutrients and drugs share common pathways and mechanisms so that, at least theoretically, acute and chronic medications (i.e. omeprazole, ezetimibe) may alter absorption, serum status, distribution, clearance and metabolism of carotenoids, and viceversa (Boullata et al, 2012). However, there is a lack of information regarding the impact of common drugs on the bioavailability and metabolism of carotenoids. Noteworthy, carotenoids can also influence the pharmacological activity of drugs by modifying their absorption and metabolizing enzyme systems. Thus, lycopene may inhibit
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ACCEPTED MANUSCRIPT CYP1A1 and CYP1B1 and increase hepatic cytochrome P4502E1 (Rodriguez-Fragoso et al, 2011). Moreover, from a clinical perspective, some interactions may be beneficial. For example, β-cryptoxanthin may interact with certain drugs acting as an enhancer and reducing the toxicity of anticancer treatments (i.e. oxaliplatin) (San Millan et al, 2015). Genetic variability. On the basis of plasma response (absorption efficiency), human volunteers were classified as responders and non-responders (“poor” and “good” absorbers) (Failla & Chitchumroonchokchai, 2005). Discrimination of subjects on the basis of plasma response has been criticized since the lack of an acute plasma response does not necessarily indicate lack of absorption, as reported in ileostomy patients
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(Faulks et al, 1997).
Both serum carotenoid status and response to supplementation have been related to genetic
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differences (single nucleotide polymorphisms (SNPs)) of several genes involved in absorption and transport of carotenoids (i.e. SR-B1, CD-36, ABC G5/G8, NPC1L1)(Lietz & Hesketh, 2009; von Lintig et
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al, 2010;Herron et al, 2006; Lietz et al, 2012; Borel et al,2012; 2014; 2015a, b). Also, SNPs in the BCMO-1 gene (encoding β-carotene 15, 15´-oxygenase) have been associated with fasting β-carotene concentrations and the ability of this enzyme to convert β-carotene into vitamin A (Leung et al, 2009)
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and SNPs in BCMO-1 and CD36 genes were associated with circulating levels of lutein, uptake of carotenoids at target tissues and the MPOD (macular pigment optical density) response to lutein supplementation (Feigl et al, 2014). Similarly, tissue concentrations of lycopene are likely dictated by
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expression and genetic variation of lipoprotein receptors, transporters and metabolizing enzymes, thus influencing lycopene accumulation at targets sites of action (Moran et al, 2013). Overall, the available evidence supports a relevant role for SNPs of carotenoid transporters and
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metabolizing enzymes as determinants of circulating levels, tissue concentrations and response,
Gut Microbiota
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although the effect may differ according to the carotenoid supplied (Wang et al, 2013).
Unabsorbed carotenoids may be active in the gastrointestinal tract as they may be also
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metabolized during intestinal transit and exert biological actions. Interestingly, animal and human studies have shown that some dietary bioactives (i.e. β-cryptoxanthin) may be absorbed through the colon where they or their metabolites (i.e. apo-carotenoids) may exert biological activity (La Frano et al,
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2013). Nevertheless, although in vitro studies suggest that at least some carotenoids (i.e. β-carotene isomers, β-cryptoxanthin esters) may be stable upon simulated fermentation conditions (Mosele et al, 2016;Hernández-Alvarez et al, 2015), the effect of microbiota and the colonic absorption remain as a major gap in the knowledge of carotenoid bioavailability (Bohn et al, 2015). 6.- CONCLUDING REMARKS. Markers of exposure and response constitute essential tools to assess bioavailability of carotenoids. Overall, both concentrations of carotenoids in different matrices and functional markers (associated biological effect) may be used both in single-dose and long-term supplementation assays. However, in general, a relationship exists between the type of marker (analytical or functional), the biological specimen needed and the time of intervention required to detect changes. Analysis of markers in matrices with rapid turnover (i.e. TRL, serum) usually reflect short-term bioavailability while cumulative exposure may be better approached by measuring carotenoids in tissues and using functional
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ACCEPTED MANUSCRIPT markers (i.e. biological effect). In any case, the validity of the studies may be compromised by methodological and biological factors including the analytical uncertainties, the timing of sample collection, pre- and post-absorptive changes, the linearity of the response and in vivo metabolism. Thus, an integrated approach including the use of combined protocols (i.e. single and multiple dose), multiple biomarkers (i.e. trans- and cis-isomers, metabolites) and biological matrices (i.e. short- and long-term turnover) may provide a more reliable estimation of carotenoid bioavailability. In addition, the potential impact of genetic variability, physiological state (i.e. subclinical inflammation), the use of drugs and gut microbiota should be also considered on assessing and interpreting the bioavailability of carotenoids in
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humans.
Conflict of Interest; None declared.
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Chapelon, F; Bingham, S; Welch, AA; Martinez-Garcia, C; Nagel, G; Linseisen, J; Quirós, JR; Peeters, PHM; van Gils, CH; Boeing, H; van Kappel, AL; Steghens JP; Riboli, E(2005) Plasma carotenoids as biomarkers of intake of fruits and vegetables: ecological-level correlations in the European Prospective
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stereoisomers. Journal ofNutrition 134, 2237-2243 Ball, GFM. (1998). Physiological aspects of vitamin bioavailability. In: Bioavailability and Analysis of vitamins in Foods. Chapman & Hall (1st ed.). Bañuls, C.; Martínez-Triguero, M.; López-Ruiz, A.; Morillas, C.; Lacomba, R.; Víctor, V.; Rocha,
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Figure legends
Figure 1.- Integrative scheme illustrating the relationships between study design, timing of sampling, biological matrix, type of biomarkers and influencing factors. This figure aims to show the relevance of combining the objective of the study, the type of intervention (short or long-term) and the
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appropriate biological matrix to measure changes in (bio) markers. Overall, short-term exposure may be approached by determining changes in analytical markers in “fast” turnover biofluids (i.e. serum, feces)
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while long-term exposure may be better evaluated by using functional tests and analytical markers in “low” turnover tissues. In both cases, interpretation of changes should be performed with caution since confounding (i.e. rate of absorption, tissue metabolism) and modulating factors (i.e. selective tissue uptake) may bias a direct relationship between markers of exposure and biological effects as surrogates
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of carotenoid bioavailability.
Table 1.- Examples of biological and health endpoints and functional (indirect) markers
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used as surrogates of carotenoid bioavailability (i.e. bioactivity). Biological end-points
Assessment Methods/ Tests
Antioxidant capacity
HPLC, ELISA, Comet assay, antioxidant enzymes activity,
(i.e. DNA damage and repair
TBARS, MDA and other lipid metabolites
Lipid /protein oxidation products) Metabolic changes and physiological changes
Insulin resistance/ glucose tolerance, lipid profile, metabolomic profile, P450 system activity, blood pressure, pulmonary function test, brachial-ankle pulse wave velocity, inflammation markers
Eye health
Dark adaptation, visual acuity, contrast sensitivity, retinal sensitivity
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Bone health
Bone mineral density, biomechanical tests, bone remodeling markers
Immune function
Cell (subpopulations) count, cytokines (IL-2, IL-1b, IFNg, TNF-a)
Gene expression/ Epigenetic changes
Up- / down regulation, -Omics (i.e. proteomic, metabolomics), Hypo- / hyper DNA methylation Microbiota profile (dysbiosis) and metabolite changes,
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Gastrointestinal health
fecal pH, variation in markers of intestinal permeability
Cognitive, behavioral and psychological function
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and inflammation
Mental performance (i.e. timed calculation,
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concentration), neuropsychological tests (i.e. 15 words learning test), cognitive performance (i.e. memory,
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psychomotor speed), behavioural tests Physical performance
Strength, endurance, muscle performance, aerobic
Skin health
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capacity, bicycle ergometer Skin color (yellowness, redness and luminance),
(i.e. beauty from within, anti-ageing)
prevention/inhibition of UVA/UVB induced oxidative
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stress and gene expression Infection challenge tests, exercise stress, glucose tolerance tests, stressor tests (i.e. mental & physical fatigue, skin irritation test)
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Table 2.- Summary of chemical forms of carotenoids potentially useful as markers for assessing short- and long-term bioavailability (i.e. exposure). In vitro systems Human Refs Species and molecular forms
Duodenal/Micellar / Caco-2
Post-prandial (TRL, Chylomicron)1
Plasma-Serum
Tissues (including breast milk)
Trans carotenes (i.e. β-carotene, lycopene, γcarotene, )
YES
YES
YES
YES
Colourless carotenes: (i.e. phytoene, phytofluene)
YES
YES
YES
CaroteneEpoxides (i.e. 5,6-epoxy-β-carotene)
NR2
Free xanthophylls (i.e. lutein, zeaxanthin, βcryptoxanthin)
YES
YES
Xanthophyll Epoxides (i.e. neoxanthin, violaxanthin, fucoxanthin, lutein-5, 6-epoxide, capsanthin5, 6-epoxide)
YES
NO
Keto-carotenoids (i.e. canthaxanthin) Hydroxi-keto-carotenoids (i.e. astaxanthin) Capsanthin, capsorubin (including cis-isomers and metabolites) Carboxi-carotenoid (i.e. bixin, norbixin) Xanthophyll esters (i.e. lutein, α- and βcryptoxanthin or astaxanthin)
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YES
Khachick et al, 1997; 2006; Moran et al, 2016; Cooperstone et al, 2015
NR
Barua et al, 1999 Meléndez-Martinez et al, 2015
YES
Khachick et al, 1997; 2006 Lipkie et al, 2015
NO (i.e. lutein-5, 6-epoxide, capsanthin-5, 6-epoxide, violaxanthin) Very low (neoxanthin, fucoxanthin)
NR
YES
YES
YES (i.e.retina)
Barua& Olson, 1991; Failla & Chitchumroonchokchai, 2005; Pérez-Gálvez et al, 2003; Granado et al, 2007; 2011; Asai et al, 2008 Sugawara et al, 2001 Kotake-Nara et al, 2011 Kistler et al, 2002; Polara-Cabrera et al, 2010
YES
YES
NR
Oshima et al, 1997; Etoh et al, 2000 Pérez-Gálvez et al, 2003
NR
YES
YES
NR
Levy et al, 1997 Polara-Cabrera et al, 2010
YES
NO
NO (Except in lutein supplemented subjects)
YES (i.e. skin)
Wingerath et al, 1995; 1998; Granado et al, 1998; Barua & Olson 2001; Failla & Chitchumroonchokchai, 2005; Breithaup et al, 2004;
NR
YES (human homonegates) NR
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YES
U N YES
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Coral-Hinostroza et al, 2004 Schlatterer et al, 2006; Burri et al, 2016 Granado et al, 2007; 2010; 2011 Cis-isomers (i.e. cis-lutein, cis-β-carotene, cis-lycopene, cis-capsanthin, cis-astaxanthin)
YES
YES (relative proportion is carotenoid-dependent)
YES (relative proportion is carotenoid-dependent)
NR
NR
Apo-carotenoids (β-apo-13-carotenone, β-apocaroten-ols & β-apo-carotenals) Lycopenoids (Apo-lycopenals) Free retinol Retinyl esters
YES
N A
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2, 6-cyclo-lycopene-diols
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Metabolites3 Meso-zeaxanthin, keto- and keto-hydroxycarotenoids (i.e. ε,ε-carotene-3-3´-dione, 3-OH-β,ε-carotene-one, 3´OHε,ε-carotene-3-one) anhydrolutein(s) 3-epi-lutein(s) capsanthone (in vivo oxidized capsanthin)
YES (Pattern tissue-specific & carotenoid dependent)
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YES (i.e. breast milk, ocular tissues) YES
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YES
Not reported
YES
NR
YES
NR
-
NO
YES (RBP) (homeostatic control)
YES (low/ very low)
-
YES
YES (<5-10% in healthy, fasted subjects)
YES (storage form)
YES
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(human homonegates)
C A NR
YES (i.e. breast milk) Not reported
Khachick et al, 1997; 2006 Lipkie et al, 2015 Moran et al, 2015; 2016; Ross et al, 2011 Etoh et al, 2004
Khachick et al, 1997; 2006 Granado et al, 1998; Bhosale et al, 2005; 2007 Thürmann et al, 2005 Etoh et al, 2004 Schlatterer et al, 2006
Khachick et al, 1997; 2006 Tang et al, 1991; Wang et al, 1992; 2015; Ho et al, 2007; Eroglu et al, 2012 Kopec et al, 2010; Goupy et al, 2012; Mein et al, 2011; Sharoni et al, 2011
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1
Detected during post-prandial period (usually 2-8h). Some studies report detection in plasma/blood during this time after dose/meal rather than prepare triglyceride-rich lipoprotein (TRL) or chylomicron fraction specifically. Some carotenoids (i.e. xanthophylls) may be transferred to other lipoproteins or tissues during this period and thus, it is assumed they are from dietary origin. 2NR; Not reported, not assessed or below detection limit of the technique employed. 3
Some compounds referred to as metabolites are also present in foods although its major contributor is thought to be in vivo metabolism.
T P
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C S U
N A
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Pharmaco-kinetic vs cumulative exposure (i.e. single vs multiple dose) Dose and duration of intervention Timing of blood collection Linearity of response / Saturation (“ceiling” effect) Compliance Type of sample (short- vs long-term exposure/ effect)
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Biological matrix
Biological variation (i.e. circadian, seasonal)
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Cell turn-over (i.e. colon and buccal mucosal cells) Pre-, analytical and post-analytical factors
Stability during sample collection, transport and storage
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Resolution and detection limit
Lack of standards (i.e. metabolites) Between methods and between labs variability (Lack of method
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Widely accepted references ranges* Cutoffs with clinical or epidemiological relevance *
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Nutrient synergism (Lack of selectivity for causal links) *
Host-related factors
Genetic variability Affecting “static” markers/response
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Affecting “functional” markers/response
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SNPs of absorption, transport and metabolism systems SNPs for enzymes and processes involved in physiological function/effects DNA methylation, up- and down-regulation
Physiological factors
Age, gender and body mass index
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Epigenetic changes /nutrigenomic actions**
Gastric pH, biliar and pancreatic secretions Intestinal absorption limits (saturation thresholds) First-pass metabolism Differential transport, transference and tissue deposition Clearance rate Tissue-specific metabolism Vitamin A status and homeostatic control Malabsorption-related disorders (i.e. diarrhea, parasites) Physiological state (i.e. inflammation) Metabolism by microbiota Diet and drug-interactions Enterohepatic recirculation?
Lifestyle factors
Tobacco, alcohol, exercise
*Factors especially relevant for functional markers /tests. ** Also dependent from other nutrients such as folate, B12, vitamin K and A& methyl group donors.
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Both host-related and methodological factors may influence static and functional markers
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Biomarkers constitute an essential tool to assess the bioavailability of carotenoids in humans. Markers for carotenoid bioavailability can be divided into direct and functional indicators. A relationship exists between the type of marker, the biological specimen and the time required for a change. Overdosing and long-term protocols may lead to saturation effects and the loss of linearity.
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