3rd Amino Acid Workshop

Biomarkers for Cancer Diagnosis: Implications for Nutritional Research1

Padma Maruvada and Sudhir Srivastava2
Cancer Biomarkers Research Group, Division of Cancer Prevention, National Cancer Institute, Bethesda, MD 20892
ABSTRACT The biology of disease progression is a complex process that involves multiple sequential steps leading to cellular changes and metabolic events. These molecular events, which may serve as potential biomarkers, can be analyzed by laboratory methods and used to detect a disease such as cancer or indicate the biological exposure to environmental substances including dietary intake. Identication of the genetic, molecular, and clinical events involved in the disease process enables the development of effective therapeutic and preventive measures and the prediction of prognostic outcomes. This article describes various factors that inuence nutritional and cancer biomarker research, draws similarities between them, and discusses the measures that have been adapted to validate cancer biomarkers that can potentially be applied to nutritional biomarker research. Nutritional research suffers from a lack of means to quantify relationships between diet and cancer. Biomarkers of dietary intake or metabolism, therefore, could have potential application in study designs for establishing a causal relationship between diet and disease. J. Nutr. 134: 1640S1645S, 2004. KEY WORDS:  biomarkers  cancer  amino acids

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Introduction The biology of disease progression is a complex process that involves multiple sequential steps leading to cellular changes, altered signaling pathways, and metabolic events. These molecular events, which may serve as potential biomarkers, can be analyzed by laboratory methods and used to detect a disease or indicate the biological exposure to environmental substances including those encountered by dietary intake. Carcinogenesis involves initiation, progression, and metastasis. Identication of the genetic, molecular, and clinical events involved in the process enables the development of targetbased therapeutic and preventive measures and the prediction of prognostic outcomes. In general, cancer biomarkers are classied based on their ability to detect the disease: early detection, high risk, prognostic, or predictive. A given marker may serve one or more of these functions and thus may fall into more than one category. For instance, prostate-specic antigen is used to detect and monitor prostate cancer in diagnosis and treatment (1). Cervical cancerspecic antigen-125 (CA-125) is commonly used for detecting cervical cancer (2). Although many of these markers help in detecting the presence of the disease, none has been validated for its usefulness in screening an asymptomatic population. Some genomic markers such as breast
1 Presented at the conference The Third Workshop on the Assessment of Adequate Intake of Dietary Amino Acids held October 2324, 2003 in Nice, France. The conference was sponsored by the International Council on Amino Acid Science. The Workshop Organizing Committee included Vernon R. Young, Yuzo Hayashi, Luc Cynober, and Motoni Kadowaki. Conference proceedings were published as a supplement to The Journal of Nutrition. Guest editors for the supplement publication were Vernon R. Young, Dennis M. Bier, Luc Cynober, Yuzo Hayashi, and Motoni Kadowaki. 2 To whom correspondence should be addressed. E-mail: srivasts@mail. nih.gov.

cancerassociated gene (BRCA)3-1 and BRCA-2 mutations identify the risk of breast cancer (3,4). Similarly, adenomatous polyposis syndrome, a genetic alteration that results in colonic polyps, helps evaluate the risk of colon cancer (5). Nutritional biomarkers, in contrast, reect the nutritional status with respect to the intake or metabolism of dietary constituents. A nutritional biomarker can be a biochemical, functional, or clinical index of status of a dietary constituent. Nutritional markers are broadly classied as short-, mid-, and long-term markers that predict the nutritional outcomes of the relevant changes in the biomarker levels to dietary changes (6). Like their diagnostic counterparts, nutritional biomarkers can also fall into more than one category depending on the intended purpose. For example, nutritional biomarkers can be used to validate dietary instruments such as dietary questionnaires. These markers may reect a direct relationship to dietary intake and can be independently assessed; they are either the dietary nutrients themselves in the body uids or direct end-products of the dietary substances. In some cases, biomarkers not only act as markers for dietary intake but also reect a measure of nutritional status for a nutrient. Such biomarkers provide useful information regarding the metabolism of the nutrient and effects of disease processes. Biomarkers and nutritional research Biomarkers are inuenced not only by disease but also by several normal physiological conditions such as age and genetic
3 Abbreviations used: BDL, biomarker development laboratory; BRCA, breast cancerassociated gene; BVL, biomarker validation laboratory; CEC, clinical and epidemiological center; EDRN, Early Detection Research Network; MALDI, matrixassisted laser desorption/ionization; SELDI, surface-enhanced laser desorption/ ionization.

0022-3166/04 $8.00 2004 American Society for Nutritional Sciences.

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and environmental factors. This is even truer in the case of nutritional inuence on the level of biomarkers. For a nutritional biomarker to be effective in nutritional research, it should reect an absolute dietary intake or the metabolic event that follows the nutrient intake. However, the specicity of a nutritional biomarker is often impeded by a number of possible interactions that occur within the body. In addition to these biological uncertainties, the choice of specimen may be critical for the assessment of a nutritional biomarker, e.g., estimation of folate in plasma is subject to dietary changes, whereas erythrocyte folate more accurately reects nutritional status (7,8). It is often intriguing to infer whether a given nutritional assessment represents the truer picture of a nutrient level, e.g., toenails and hair are considered ideal surrogates for selenium estimation; however, the validity of these markers has been questioned (6,9). In many instances, the robustness of an analytical technique is a critical factor. For example, analysis of vitamin C is a problematic issue for population studies, because the timely processing of the samples and the requirement of freshly prepared buffer for storage are of paramount importance (9). It is unclear whether one biomarker could reect nutritional status or whether a combination of biomarkers is needed, because it is often suggested that the actual protective effect of nutrients against diseases is the intake of a wide array of nutrients (10). Nutritional biomarker evaluation thus requires an extensive systematic approach to establish the status of the population, and the associated statistical and epidemiologic parameters are critical for nutritional research. In this article, we describe various factors that inuence nutritional and cancer biomarker research and draw similarities between them. Moreover, we also discuss measures that have been adapted to validate cancer biomarkers that can potentially be applied to nutritional biomarker research. Biological aspects. The biological relevance of a nutritional marker can only be ascertained after confounding factors are controlled, which requires a comprehensive understanding of the biology and analytical measurements. A nutrient of interest may be in a mixture of compounds with similar biological action, e.g., endocrine disrupters or antioxidants. In such cases, the total content of these compounds rather than a single nutrient should be measured (11). Similarly, there are ;50 types of carotenoids consumed in the human diet, whereas only four or ve compounds are found in serum in measurable amounts, and b-carotene is the only one measured (12,13). In addition, the biomarker of interest may be the metabolite of a dietary compound that is responsible for the biological effect, which makes the biomarker research more complex. Adding to the complexity, the bioavailability of a nutritional biomarker can be affected by other dietary factors such as fat, carotenoids, or host factors, which may modify the correlation between the level of nutrient intake and the biomarker (11,14,15). Many dietary substances such as amino acids, when consumed in larger quantities, were found to have adverse effects on experimental animals (16). However, it is difcult to establish such toxic effects in humans because of the lack of quantitative data. One of the major problems associated with using biomarkers is that biomarkers vary with age, lifestyle, and environmental factors. For this reason it is essential to develop methods that can selectively detect changes in the biomarker level that are related to exposure and not to other environmental or physiological factors, especially for a wide range of study designs including aggregate (ecological) and analytical observational study designs and randomized control trials (17).

There is variation among individuals in physiology and nutrient metabolism. The absorption of a nutrient by the body depends on the nutritional status of the tissues. Low calcium or iron levels would prompt efcient absorption across the lumen (18,19). Similarly, the presence of certain substances in the diet may interfere with absorption, as in the case of phytic or oxalic acids, which interfere with divalent ion absorption (18). Absorption of vitamin B-12, for example, is largely inuenced by the presence of intestinal intrinsic factor, and the actual deciency of the vitamin is attributed to the absence of this factor (20). The presence of certain compounds in the diet as both contaminants and additives may determine the etiology of the disease and thus may need to be evaluated. Food processing may incorporate several toxic or benecial bioactive substances that need to be continuously monitored (8). Corollary between diagnostic and nutritional biomarkers. Biomarkers for cancer detection and biomarkers for nutritional research share several common attributes and performance characteristics, although the signicance may vary with the nal outcome. In the case of diagnostic markers, the nal outcome is always the incidence of disease; however, in the nutritional setting, the outcome/use varies, e.g., level of nutrients, level of intake, etc. (Table 1). These performance characteristics must be addressed before they are applied to population studies. Sensitivity. Sensitivity is dened as the true-positive test results (individuals with disease who test positive for that disease) expressed as a percentage of all tested individuals who have that disease (total of true positives and false negatives). When applied to a disease such as cancer, sensitivity is the chance that a person with the disease will have a positive test. Specicity. Specicity describes the true-negative results (tested individuals without disease who test negative for that disease) expressed as a percentage of all tested individuals who do not have that disease (the total of true negatives and false positives). When applied to cancer, specicity is the chance that a person without cancer will have a negative test. Positive predictive value. The positive predictive value is the proportion of individuals who test positive and also have the disease. The requirements for the performance characteristics of a biomarker vary with its intended use. For diagnosis or monitoring, high sensitivity is important, whereas for screening, specicity is of paramount importance. However, in some cases, for example, in hepatitis C viral infection, sensitivity is more important for screening (21). Sensitivity and specicity have different interpretations in the context of nutritional research. For example, in the context of nutritional uptake, sensitivity refers to the ability of an assay to measure the true concentrations of a nutrient of interest independent of confounding variables such as absorption and metabolic rates, age, sex, etc., whereas specicity refers to the ability of an assay to distinguish the nutrient of interest from other nutrients. If the purpose of the biomarker is to stratify subjects based on their basal level from an altered level of a nutrient, then the sensitivity and specicity have requirements similar to their diagnostic counterparts. Here the objective is to minimize classication error rates in reference to a particular nutrient. The ability to measure the concentration of a nutrient in body uids may not reect much about the ability to measure the nutrient intake. To alleviate the problems associated with a biomarker of exposure such as toxicity related to amino acid intake, either the sensitivity of the assay must increase, or a large study population is required to detect differences between the physiological basal level and an increased intake of the amino acids.

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TABLE 1
Performance characteristics of nutritional and diagnostic biomarkers1
Parameter Measurement error Precision Performance characteristics Validity
1

Nutritional biomarker Preanalytical, analytical, misclassication Technique, reagents, calibration, instrumentation Sensitivity, specicity, predictive values, varied outcome Correlation with true nutritional outcome

Diagnostic biomarker Similar to nutritional biomarker Similar to nutritional biomarker Similar to nutritional biomarker; outcome with disease incidence Correlation with true disease outcome

Nutritional and diagnostic markers share similar challenges and inuences.

Methodological aspects. Although a variety of specimens can be selected for identifying biomarkers, specimens collected by noninvasive means are more acceptable and easier to handle and store. A selected specimen should contain ranges of biomarkers that are detectable by laboratory methods and be physiologically relevant. Such specimens could include blood, urine, stool, and sputum. However, the choice of specimen depends on the purpose of the study. For example, hair, nails, and breast milk may serve as better specimens for analyzing some biomarkers such as trace elements (9). Certain specimens such as fecal samples may only be amenable for small-scale studies, whereas some of the exhaustive sample-collection procedures such as 24-h urine-sample collection for dietary nitrogen may be required for some studies. Systemic biomarkers such as hormones and other regulators of metabolism may predict overall health status pertaining to that nutrient, whereas more specic tissue-related biomarkers such as bonedensity to predict the onset of osteoporosis might provide information on organ development and response to nutritional status (22). Laboratory issues. Proper sample handling and storage are of utmost importance when analyzing biomarkers. Urine, for example, may need to be acidied to improve the shelife of the samples. Some biomarkers may be unstable when exposed to light, heat, oxygen, or other factors or may simply decay over time. Care must be taken to avoid exposure of the samples to a variety of laboratory conditions, and the amount of time elapsed between the sample collection and analysis may be critical. The assay to detect a biomarker should be relatively simple and cost effective and should have the capacity for highthroughput scalability. Appropriate quality-control ranges must be present whenever possible. Success of an analytical method relies on the sensitivity of the method to detect the biomarker in samples at minimum levels or in repeated analysis. For this reason, the validity, reliability, and repeatability of the results of the biomarker assay are of great importance. Repeatability refers to the sensitivity of the analytical method to detect the biomarker in question with repeated measurements. Reliability refers to the correlation of measurements for a biomarker in repeated assays. Validity of the biomarker refers to the ability to identify a highly repeatable characteristic of a biological sample. It is usually described as the correspondence of a biomarker with the actual exposure. Some samples may render themselves as informative tools for studying certain nutritional issues but may not be applicable to larger studies. For example, fecal components can be important for measuring bers or bile acids that are relevant to colon cancer and other diseases; however, they can only be applied to small-scale studies. Urine samples may be valuable to study water-soluble nutrients but are limited by the nutrient saturation of the tissues and dietary intake (6). Some samples like breast milk may be easily accessible but may not be relevant to many diseases with epidemiological

importance. Knowledge of the presence of the biomarker in a given serologic component is essential so that the appropriate component can be collected without collecting all the fractions for the analysis. Technological aspects. Current advances in genomics, epigenomics, proteomics, and metabolomics have greatly accelerated biomarker discovery for nutritional research. By determining molecular patterns under various conditions, it is possible to develop a molecular signature for a given disease or physiological status. Also, these proles could be used to measure the inuence of dietary components. These novel approaches largely complement the conventional biochemical or imaging methods in identifying more effective biomarkers. Approaches to biomarker discovery Several approaches specic to molecular events have emerged for biomarker discovery. These are briey summarized here. Nutrigenomics. Determination of molecular patterns under various nutritional conditions can yield interesting results related to the genes that are turned on or off in pathways related to nutrient metabolism. The techniques of transcriptomics include DNA-based large-scale microarrays, differential display, and serial analysis of gene expression to study gene-expression patterns in response to external stimuli or nutrients (23). Recently it was shown that amino acids can modify the gene expression of target genes (24). Amino acid depletion could trigger many molecular events leading to the activation of upregulation and translation of the cationic amino acid transporter gene (25). In rat hepatocytes, addition of amino acids leads to swelling, which implies regulation of gene expression (26,27). Nutritional proteomics. Although gene-expression patterns provide substantial information about the physiological response to external nutrients, they do not always correlate with protein concentrations. Moreover, proteins are subject to proteolysis or post-translational modications such as glycosylation or phosphorylation that are not evident from genomicbased biomarkers. Biomarker discovery strategies that target protein expression are becoming popular because proteomic approaches enable the characterization of proteins involved in disease progression or physiological conditions. Various proteomic approaches include two-dimensional electrophoresis, isotope-coded afnity tags, surface-enhanced laser desorption/ ionization (SELDI), matrix-assisted laser desorption/ionization (MALDI), liquid chromatography coupled to mass/mass spectroscopy, and MALDItime of ight; these methods give rise to protein ngerprints, tissue-based arrays, protein microarrays, and antibody-based arrays. Although the eld is still evolving, there is great promise for novel biomarker development. In a recent study, Sironi et al. (28) used proteomic approaches

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to show the effects of salt loading on the protein patterns in body uids. Van Eijk and Deutz (29) studied the plasma proteinsynthesis patterns using proteomic approaches. Using a targeted proteomic approach, Brooks et al. (17) showed that the angiotensin II type 1a receptor plays an important role in regulation of Na1 transporter and channel proteins. Nutritional metabolomics. A general limitation of expression studies is the correlation between the changes in gene expression to the functional assignment of specic genes. Metabolic changes inuence nutritional requirements and utilization. Examining metabolomics or changes in metabolic proles can be an important part of an integrative approach for assessing physiological responses to external agents. A comprehensive approach is important in identifying a nutritional biomarker, because the biomarkers of nutritional status relate to changes in a wide variety of trace elements, vitamins, and other nonorganic or small organic molecules. Recent metabolomic advances include nuclear magnetic resonance spectroscopy, mass spectrometry, chromatographic analysis, and metabolic network analysis models to predict metabolic uxes. Xu et al. (30) showed that peroxisome proliferatoractivated receptor-a inuences substrate utilization for glucose production in hepatocytes. Rosiglitazone has been shown to induce varied effects on tissue metabolism and the lipid metabolome (31). Although systemic metabolomic signatures can be more informative, organ-specic metabolomic approaches would reduce the complexity and yield helpful information about dietary factors (32). Nutritional epigenomics. Certain environmental factors inuence the selective activation or inactivation of a gene or a set of genes by epigenetic changes such as methylation of DNA patterns, mostly in the promoter region, or acetylation of histones (33). In many events these patterns are inherited, which makes them vital targets for disease progression. It was shown that dietary substances bring about many of these epigenetic changes by affecting DNA methylation through changing the availability of methylating compounds such as S-adenosylmethionine or inuencing methylating pathways (34). For this reason, exploration of epigenetic nutritional biomarkers may give rise to helpful information about the roles of dietary substances in relation to diseases such as cancer. Challenges with nutritional biomarkers Biomarkers offer a tremendous amount of information about health and disease and therefore hold great promise for nutritional epidemiology. However, the biomarker evaluation is often impeded by a lack of ideal endpoints and suitable laboratory methods to detect these conditions. Although signicant progress can be achieved in this eld through animal models, unfortunately, good models are still lacking for the study of various nutritional conditions. Other issues that pertain to nutritional biomarkers as discovery pathways include a lack of knowledge of the dietary constituents that may play a signicant role in health and disease and uncertainties due to the dynamic nature of dietary changes and the difculties in quantifying them effectively. Clearly, there is a demand for nutritional biomarker research that can address these issues. Technological advances add another confounding factor to these uncertainties. Although our knowledge of biomarkers has expanded over the years, assays and analyses based on genomic and proteomic approaches have become increasingly challenging. From a practical viewpoint, these assays need to be robust and thereby lend themselves as powerful tools to biomarker research. However, such approaches are successful only if the

procedures are simple, inexpensive, and minimally invasive. Development of high-throughput assays is highly desirable to allow us to screen larger populations in less time. Biomarkers based on one technology may not necessarily provide all the needed information regarding physiological status. Similarly, DNA-sequence data are limited by a lack of information on post-translational modications or biological consequences of altered gene expression due to environmental factors such as diet. We still lack effective computer models to simulate various physiological conditions and thereby provide a wealth of information without the need for expensive, long-term clinical trials and experimental research. These simulation studies could provide information on the natural history of the nutritional status and the biomarker, treatment effects on the nutritional status in relation to disease, cost information, and population behavior (3537). Bioinformatics must evolve to enable the analysis and integration of the high-dimensional, high-throughput data that are generated on such multivariate platforms. Many technologies such as proteomics and metabolomics are still in their infancy and therefore necessitate a systematic iterative approach for biomarker research. Cancer biomarker research suffers from a lack of validated biomarkers that can successfully predict risk and detect the disease at an early stage and thereby improve the chances of therapeutic intervention. Although many efforts have been focused on biomarker discovery leading to a wide range of promising biomarkers, none has been validated. Unfortunately there are no recommended criteria for validating a biomarker, although some biomarker approaches (such as Pap-smear testing to screen for cervical cancer) have gained wider acceptance on their own over time (38,39). In an effort to accelerate biomarker research, the National Cancer Institutes Early Detection Research Network (EDRN) has adapted a ve-phase approach to biomarker development and evaluation (40). The purpose of the EDRN is to coordinate research among biomarker development laboratories, biomarker validation laboratories, clinical repositories, and population-screening programs. By coordinating these efforts, EDRN hopes to facilitate collaboration and promote quality and timely efforts in cancer biomarker development. Currently there are ;18 biomarker development laboratories (BDLs), 2 biomarker validation laboratories (BVLs), and 8 clinical and epidemiological centers (CECs) that are constantly monitored by the Data Management and Coordination Center, which supports the infrastructure of EDRN (Fig. 1). To meet the goals of EDRN, ve phases of biomarker development have been proposed. Phase I: Preclinical exploratory studies to identify putative biomarkers that are unique to cancer. Researchers at BDLs focus their efforts on identifying biomarkers on various technological platforms, especially noninvasive assays that are easy to adapt across the laboratories, which makes them more affordable. The biomarker data are evaluated for functional characteristics to ascertain how well the biomarker distinguishes the control from the cases. Phase II: Clinical assay and validation to distinguish patients with cancer from those without the disease based on the presence or absence of the biomarker in the study and to determine biomarker sensitivity and selectivity. Once a putative biomarker is identied, BVL researchers develop a suitable clinical assay to distinguish the cancer cases from controls based on the biomarker data. The assay is evaluated for robustness and statistical properties apart from optimizing its performance. This also provides

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Phase IV: Prospective screening. Screening is conducted to determine the operating characteristics of the biomarkerbased screening test in a relevant population by determining the detection and false-referral rates. Other details such as the practical feasibility of implementing the screening program and the compliance of screenpositive subjects with workup and treatment recommendations are worked out in this phase. Phase V: Cancer control study. Controlled studies are conducted to estimate the reductions in cancer mortality rate afforded by the screening test. Additionally, information about screening and treatment costs and compliance with the screening and workup in a diverse range of settings are evaluated and compared with other screening programs with regard to mortality rates and costs. These phases are being successfully applied at EDRN to decide which biomarker is worthy of clinical validation using a triage system as depicted in Figure 2. Since its conception in 1999, many biomarkers in the pipeline are being validated for potential application in cancer detection. For example, microsatellite-instability assay is under validation for its potential application in bladder cancer diagnosis. SELDI proteomic signatures are being evaluated for detection of prostate cancer. A similar approach can also be adapted to develop nutritional biomarkers for epidemiological studies wherever applicable. The actual priorities can be modied based on the specic objectives of the nutritional status being studied. Nutritional biomarkers must be thoroughly evaluated for efcacy and properties. Establishment of scientic consortia such as EDRN for nutritional biomarker development may facilitate coordination among researchers for biomarker development and validation and for promotion of the process of nutritional biomarker development. In addition, smaller, welldened, controlled studies should be conducted with newer markers before larger population studies are addressed. Although no single method of biomarker evaluation is perfect, a combination of efforts is required to validate the usefulness of nutritional biomarkers.

FIGURE 1 Organization of the EDRN. BDLs, BVLs, and clinical centers are managed by a steering committee, which is in turn advised by the advisory committee and supported by data management and the coordination center.

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an opportunity to understand the relationship between biomarker measurements made on tissues and those made on noninvasive clinical specimens. Furthermore, other inuencing factors such as age, sex, and behavioral patterns on the biomarker status are determined against matched controls. Phase III: Retrospective longitudinal repository studies. Studies are conducted to evaluate the biomarker for its ability to detect the preclinical disease and dene the criteria for a positive screening test at CECs. Other covariates on the discriminatory power of the biomarker before clinical diagnosis such as demographics and other disease-related characteristics are explored with development of algorithms for screening positivity based on the marker.

FIGURE 2 Application of biomarkers from laboratory to clinic through EDRN. Biomarkers that arise from various molecular studies are evaluated in BDLs for the ability to detect cancer. Statistical performance characteristics are determined in limited samples or using low-throughput assays. Promising biomarkers are channeled through BVLs, where performance characteristics and ability to discriminate normal cases from cancer are determined apart from the development of high-throughput, reliable assays for the biomarkers. The biomarkers or assays are evaluated for their quality control (Q/C) and quality assurance (Q/A) aspects and for Clinical Laboratory Improvement Amendment (CLIA) categorization for commercialization. The validated biomarkers are tested further for their discriminatory power using longitudinal studies in a protocoldriven manner at the CECs before they are qualied as authentic biomarkers for early cancer detection.

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Future directions Biomarkers in health and disease could provide useful information for nutritional studies. However, it is often difcult to establish the causality between health states and specic dietary constituents in the rst place. To successfully interpret nutritional exposure and outcome, it is necessary to assess conditions with a near-perfect biomarker that can evaluate nutritional status and external factors effectively. It may be possible to address the issue in an iterative manner wherein biomarkers may help to reveal more about mechanisms, which in turn may assist in the development of other biomarkers. The iterative process allows for successful accumulation of information on biomarkers. Potential success of a biomarker depends essentially on its sensitivity, specicity, predictive value, ease of use, applicability, and relevance. Ultimately the true value of a biomarker depends on how much it can be used to measure the impact of diet on health. The link between diet and health, which can be objectively measured through continuous efforts in biomarker research, will open new doors to disease prevention and cure. LITERATURE CITED
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