Development and Evaluation of Active Case Detection Methods to Support Visceral Leishmaniasis Elimination in India.

As India moves toward the elimination of visceral leishmaniasis (VL) as a public health problem, comprehensive timely case detection has become increasingly important, in order to reduce the period of infectivity and control outbreaks. During the 2000s, localized research studies suggested that a large percentage of VL cases were never reported in government data. However, assessments conducted from 2013 to 2015 indicated that 85% or more of confirmed cases were eventually captured and reported in surveillance data, albeit with significant delays before diagnosis. Based on methods developed during these assessments, the CARE India team evolved new strategies for active case detection (ACD), applicable at large scale while being sufficiently effective in reducing time to diagnosis. Active case searches are triggered by the report of a confirmed VL case, and comprise two major search mechanisms: 1) case identification based on the index case's knowledge of other known VL cases and searches in nearby houses (snowballing); and 2) sustained contact over time with a range of private providers, both formal and informal. Simultaneously, house-to-house searches were conducted in 142 villages of 47 blocks during this period. We analyzed data from 5030 VL patients reported in Bihar from January 2018 through July 2019. Of these 3033 were detected passively and 1997 via ACD (15 (0.8%) via house-to-house and 1982 (99.2%) by light touch ACD methods). We constructed multinomial logistic regression models comparing time intervals to diagnosis (30-59, 60-89 and ≥90 days with <30 days as the referent). ACD and younger age were associated with shorter time to diagnosis, while male sex and HIV infection were associated with longer illness durations. The advantage of ACD over PCD was more marked for longer illness durations: the adjusted odds ratios for having illness durations of 30-59, 60-89 and >=90 days compared to the referent of <30 days for ACD vs PCD were 0.88, 0.56 and 0.42 respectively. These ACD strategies not only reduce time to diagnosis, and thus risk of transmission, but also ensure that there is a double check on the proportion of cases actually getting captured. Such a process can supplement passive case detection efforts that must go on, possibly perpetually, even after elimination as a public health problem is achieved.

Morin as an imminent functional food ingredient: an update on its enhanced efficacy in the treatment and prevention of metabolic syndromes.

Flavonoids represent polyphenolic plant secondary metabolites with a general structure of a 15-carbon skeleton comprising two phenyl rings and a heterocyclic ring. Over 5000 natural flavonoids (flavanones, flavanonols, and flavans) from various plants have been characterized. Several studies provide novel and promising insights into morin hydrate for its different biological activities against a series of metabolic syndromes. The present review is a rendition of its sources, chemistry, functional potency, and protective effects on metabolic syndromes ranging from cancer to brain injury. Most importantly this systematic review article also highlights the mechanisms of interest to morin-mediated management of metabolic disorders. The key mechanisms (anti-oxidative and anti-inflammatory) responsible for its therapeutic potential are well featured after collating the in vitro and in vivo study reports. As a whole, based on the prevailing information rationalizing its medicinal use, morin can be identified as a therapeutic agent for the expansion of human health.

Should 'Omics' education be a part of allied health profession curricula?

Sequencing of human genome followed by monumental progress in omics sciences within last two decades has made personalized nutrition for better health is a reality for near future. The complexity of underlying science in making personalized nutrition recommendation has led to the need for training of health care providers. The International Society of Nutrigenetics/Nutrigenomics (ISNN) has mission to increase the understanding among both professionals and the general public of the role of genetic variation and nutrients in gene expression. To bring this mission to fruition, we need trained healthcare professionals ready to educate public. With this in mind, we have surveyed allied health students for their omics knowledge, desire to learn more and their perception of the need of omics education. The results show a need for training in omics in all allied health disciplines and desire of the students to learn more.

Advanced Glycation End Products and Risks for Chronic Diseases: Intervening Through Lifestyle Modification.

Advanced glycation end products (AGEs) are a family of compounds of diverse chemical nature that are the products of nonenzymatic reactions between reducing sugars and proteins, lipids, or nucleic acids. AGEs bind to one or more of their multiple receptors (RAGE) found on a variety of cell types and elicit an array of biologic responses. In this review, we have summarized the data on the nature of AGEs and issues associated with their measurements, their receptors, and changes in their expression under different physiologic and disease states. Last, we have used this information to prescribe lifestyle choices to modulate AGE-RAGE cycle for better health.

"Omics" Education in Dietetic Curricula: A Comparison between Two Institutions in the USA and Mexico.

BACKGROUND/AIMS: The completion of sequencing of the human genome and a better understanding of epigenomic regulation of gene expression have opened the possibility of personalized nutrition in the near future. This has also created an immediate need for trained personnel qualified to administer personalized nutrition education. Of all the allied healthcare personnel, dietitians are the most likely to undertake this role. However, dietitians and dietetic students are still deficient in their knowledge of nutrigenomics and other "omics" technologies. Therefore, with the eventual goal of dietetic curriculum reorganization, the International Society of Nutrigenetics/Nutrigenomics (ISNN) has set out to evaluate nutrigenomic knowledge among dietetic students from different countries. In this study, we compared nutrition and dietetic students from Texas Woman's University (TWU) and the Universidad Autónoma de Nuevo León (UANL) for their perceived need for, interest in, and knowledge of different topics within nutritional genomics. METHODS: Students from both universities were sent an e-mail link to the survey which was located at psychdata.com. One hundred twenty-seven students completed the survey. The survey assessed the students' knowledge of, perceived need for, and interest in different omics technologies, as well as their basic knowledge of basic nutrition and genetic topics. Differences were assessed using the χ2 test for homogeneity and Fisher's exact test. RESULTS: Students from TWU and UANL exhibited differences in their knowledge, desire to learn more, and perceived need for omics science in some but not all categories. CONCLUSIONS: Undergraduate nutrition students from both the USA and Mexico lack a high level of knowledge in different omics topics but recognize the role that omics will play in their future as dietitians. There were differences between the 2 universities in terms of the desire to learn more about different omics technologies and to take more classes covering different topics with nutritional genomic components. In order to make personalized nutrition a reality, future dietitians will need to become fluent in different omics technologies.

Guide for Current Nutrigenetic, Nutrigenomic, and Nutriepigenetic Approaches for Precision Nutrition Involving the Prevention and Management of Chronic Diseases Associated with Obesity.

Chronic diseases, including obesity, are major causes of morbidity and mortality in most countries. The adverse impacts of obesity and associated comorbidities on health remain a major concern due to the lack of effective interventions for prevention and management. Precision nutrition is an emerging therapeutic approach that takes into account an individual's genetic and epigenetic information, as well as age, gender, or particular physiopathological status. Advances in genomic sciences are contributing to a better understanding of the role of genetic variants and epigenetic signatures as well as gene expression patterns in the development of diverse chronic conditions, and how they may modify therapeutic responses. This knowledge has led to the search for genetic and epigenetic biomarkers to predict the risk of developing chronic diseases and personalizing their prevention and treatment. Additionally, original nutritional interventions based on nutrients and bioactive dietary compounds that can modify epigenetic marks and gene expression have been implemented. Although caution must be exercised, these scientific insights are paving the way for the design of innovative strategies for the control of chronic diseases accompanying obesity. This document provides a number of examples of the huge potential of understanding nutrigenetic, nutrigenomic, and nutriepigenetic roles in precision nutrition.

Association between serum uric acid and metabolic syndrome components in prepubertal obese children (Tanner Stage I) from Nuevo León, Mexico - a preliminary study.

BACKGROUND: Metabolic syndrome (MetS) is a major risk factor for cardiovascular disease and diabetes. Previous studies in obese children demonstrating a positive association between serum uric acid (sUA) and components of MetS are confounded by lack of uniformity in age and pubertal status of children. Therefore, we have examined the role of sUA in MetS and its components in pre-pubertal children (Tanner Stage I, age ≤ 9 years). METHODS: Pre-pubertal obese children (32 boys, 27 girls, age 6-9 years) were recruited from Nuevo Leon, Mexico. For comparison, an equal number of children with normal body mass index (BMI) in the same age range (22 Boys, 39 girls, age 6-9 years) were also recruited from the same community. Presence of MetS and its components was defined according to the criteria of International Diabetes Federation. Fasting blood was analyzed for lipids, glucose, insulin, and uric acid. RESULTS: Among the obese children, sUA was positively associated with insulin resistance and hypertriglyceridemia and negatively associated with high density lipoprotein-cholesterol (HDLc). Subjects were three times more likely to have a MetS diagnosis per one unit (md/dL) difference in sUA. Of the 59 obese pre-pubertal children, 20 were classified as having MetS defined by the presence of abdominal obesity and two or more of other components described under methods. Of these, 57.1% (20/61) had sUA between 5.1 and 7.1 mg/dl. CONCLUSIONS: The findings of this study clearly indicate a positive relationship between uric acid and MetS and its components in pre-pubertal obese children with Tanner stage I and ≤9 years of age.

Assessment of anti-cancerous potential of 6-gingerol (Tongling White Ginger) and its synergy with drugs on human cervical adenocarcinoma cells.

The anti-cancerous activity of 6-gingerol extracted from Tongling White Ginger was investigated. 6-Gingerol inhibited the growth of HeLa cells with IC50 (96.32 µM) and IC80 (133.01 µM) and led to morphological changes, induced the cell cycle arrest in G0/G1-phase and ultimately resulted into apoptosis. Among cell cycle-related genes and proteins, the expression of cyclin (A, D1, E1) reduced, while of CDK-1, p21 and p27 showed slight decrease, except cyclin B1 and E1 (protein). Western blotting reported the induction of apoptosis with an increased Bax/Bcl-2 ratio, release of cytochrome c, cleavage of caspase-3, -8, -9 and PRPP in treated cells. 6-Gingerol activated AMPK, but inhibited PI3K/AKT phosphorylation with reduced P70S6K expression and also suppressed the mTOR phosphorylation. 6-Gingerol with 5-FU and Ptx resulted in 83.2% and 52% inhibition respectively, this synergy have stimulated apoptosis proteins more efficiently as compared to 6-Gingerol alone (10.75%) under in vitro conditions.

Resistant starch lowers postprandial glucose and leptin in overweight adults consuming a moderate-to-high-fat diet: a randomized-controlled trial.

BACKGROUND: High-amylose maize resistant starch type 2 (HAM-RS2) stimulates gut-derived satiety peptides and reduces adiposity in animals. Human studies have not supported these findings despite improvements in glucose homeostasis and insulin sensitivity after HAM-RS2 intake which can lower adiposity-related disease risk. The primary objective of this study was to evaluate the impact of HAM-RS2 consumption on blood glucose homeostasis in overweight, healthy adults. We also examined changes in biomarkers of satiety (glucagon-like peptide-1 [GLP-1], peptide YY [PYY], and leptin) and body composition determined by anthropometrics and dual-energy x-ray absorptiometry, dietary intake, and subjective satiety measured by a visual analogue scale following HAM-RS2 consumption. METHODS: Using a randomized-controlled, parallel-arm, double-blind design, 18 overweight, healthy adults consumed either muffins enriched with 30 g HAM-RS2 (n = 11) or 0 g HAM-RS2 (control; n = 7) daily for 6 weeks. The HAM-RS2 and control muffins were similar in total calories and available carbohydrate. RESULTS: At baseline, total PYY concentrations were significantly higher 120 min following the consumption of study muffins in the HAM-RS2 group than control group (P = 0.043). Within the HAM-RS2 group, the area under the curve (AUC) glucose (P = 0.028), AUC leptin (P = 0.022), and postprandial 120-min leptin (P = 0.028) decreased independent of changes in body composition or overall energy intake at the end of 6 weeks. Fasting total PYY increased (P = 0.033) in the HAM-RS2 group, but changes in insulin or total GLP-1 were not observed. Mean overall change in subjective satiety score did not correlate with mean AUC biomarker changes suggesting the satiety peptides did not elicit a satiation response or change in overall total caloric intake. The metabolic response from HAM-RS2 occurred despite the habitual intake of a moderate-to-high-fat diet (mean range 34.5% to 39.4% of total calories). CONCLUSION: Consuming 30 g HAM-RS2 daily for 6 weeks can improve glucose homeostasis, lower leptin concentrations, and increase fasting PYY in healthy overweight adults without impacting body composition and may aid in the prevention of chronic disease. However, between-group differences in biomarkers were not observed and future research is warranted before specific recommendations can be made. TRIAL REGISTRATION: None.

The rheological properties of polysaccharides sequentially extracted from peony seed dreg.

The peony seed dreg polysaccharides (PSDPs) were sequentially extracted using hot buffer (HBSS), chelating agent (CHSS), dilute alkaline (DASS) and concentrated alkaline (CASS). The rheological properties of PSDPs were investigated by steady-shear and oscillatory rheological measurements. The four PSDPs fractions in solution exhibited typical non-Newtonian and shear-thinning behavior. The viscosity of HBSS was higher than the rest. While the viscosity value of all PSDPs solution decreased at acid pH (4.0) and alkaline pH (10.0), in the presence of Ca(2+) and high temperature (90°C), it increased in the presence of Na(+) and following freezing. The modulus G' and G" of all PSDPs solution were increased with increasing oscillation frequency ranging between 0.01 and 100Hz at each concentration. In all four cases, the crossover of G' and G" values decreased gradually with increasing concentration of samples.

Guide and Position of the International Society of Nutrigenetics/Nutrigenomics on Personalized Nutrition: Part 2 - Ethics, Challenges and Endeavors of Precision Nutrition.

Nutrigenetics considers the influence of individual genetic variation on differences in response to dietary components, nutrient requirements and predisposition to disease. Nutrigenomics involves the study of interactions between the genome and diet, including how nutrients affect the transcription and translation process plus subsequent proteomic and metabolomic changes, and also differences in response to dietary factors based on the individual genetic makeup. Personalized characteristics such as age, gender, physical activity, physiological state and social status, and special conditions such as pregnancy and risk of disease can inform dietary advice that more closely meets individual needs. Precision nutrition has a promising future in treating the individual according to their phenotype and genetic characteristics, aimed at both the treatment and prevention of disease. However, many aspects are still in progress and remain as challenges for the future of nutrition. The integration of the human genotype and microbiome needs to be better understood. Further advances in data interpretation tools are also necessary, so that information obtained through newer tests and technologies can be properly transferred to consumers. Indeed, precision nutrition will integrate genetic data with phenotypical, social, cultural and personal preferences and lifestyles matters to provide a more individual nutrition, but considering public health perspectives, where ethical, legal and policy aspects need to be defined and implemented.

Guide and Position of the International Society of Nutrigenetics/Nutrigenomics on Personalised Nutrition: Part 1 - Fields of Precision Nutrition.

Diversity in the genetic profile between individuals and specific ethnic groups affects nutrient requirements, metabolism and response to nutritional and dietary interventions. Indeed, individuals respond differently to lifestyle interventions (diet, physical activity, smoking, etc.). The sequencing of the human genome and subsequent increased knowledge regarding human genetic variation is contributing to the emergence of personalized nutrition. These advances in genetic science are raising numerous questions regarding the mode that precision nutrition can contribute solutions to emerging problems in public health, by reducing the risk and prevalence of nutrition-related diseases. Current views on personalized nutrition encompass omics technologies (nutrigenomics, transcriptomics, epigenomics, foodomics, metabolomics, metagenomics, etc.), functional food development and challenges related to legal and ethical aspects, application in clinical practice, and population scope, in terms of guidelines and epidemiological factors. In this context, precision nutrition can be considered as occurring at three levels: (1) conventional nutrition based on general guidelines for population groups by age, gender and social determinants; (2) individualized nutrition that adds phenotypic information about the person's current nutritional status (e.g. anthropometry, biochemical and metabolic analysis, physical activity, among others), and (3) genotype-directed nutrition based on rare or common gene variation. Research and appropriate translation into medical practice and dietary recommendations must be based on a solid foundation of knowledge derived from studies on nutrigenetics and nutrigenomics. A scientific society, such as the International Society of Nutrigenetics/Nutrigenomics (ISNN), internationally devoted to the study of nutrigenetics/nutrigenomics, can indeed serve the commendable roles of (1) promoting science and favoring scientific communication and (2) permanently working as a 'clearing house' to prevent disqualifying logical jumps, correct or stop unwarranted claims, and prevent the creation of unwarranted expectations in patients and in the general public. In this statement, we are focusing on the scientific aspects of disciplines covering nutrigenetics and nutrigenomics issues. Genetic screening and the ethical, legal, social and economic aspects will be dealt with in subsequent statements of the Society.

Physicochemical properties and antioxidant activities of polysaccharides sequentially extracted from peony seed dreg.

The sequential extraction of peony seed dreg polysaccharides (PSDP) with hot buffer (HBSS), chelating agent (CHSS), dilute alkaline (DASS) and concentrated alkaline (CASS) yielded four different polysaccharide fractions. Based on their absorptions at 3600-3200cm(-1) and 1200-800cm(-1), these fractions were confirmed to be polysaccharides. The properties of four PSDPs displayed some slight differences. The CASS showed the highest peak temperature and endothermic enthalpy. The emulsifying activity and emulsifying stability of four PSDPs exhibited a dose-dependent pattern; HBSS showed the highest emulsifying activity, and CHSS displayed the longest emulsifying stability. The four PSDPs also exhibited wide variations in their antioxidant activities. For example, i) CASS showed the highest DPPH radical scavenging activity, reducing power and ABTS radical scavenging activity; ii) HBSS exhibited the highest hydroxyl radical scavenging activity, and iii) CHSS displayed the higher ferrous ions chelating ability than others.

Advanced Glycation End Products, Inflammation, and Chronic Metabolic Diseases: Links in a Chain?

Advanced glycation end products (AGEs) are a diverse group of compounds produced when reducing sugars react with proteins or other compounds to form glycosylated molecules. AGEs may form endogenously, and glycation of molecules may negatively affect their function. AGEs may also be consumed in food form with dietary AGEs reported to be particularly high in foods treated with high heat: baked, broiled, grilled, and fried foods. Whether dietary AGEs are absorbed in significant quantities and whether they are harmful if absorbed is a question under current debate. The American Diabetes Association makes no recommendation regarding avoidance of these foods, but many researchers are concerned that they may be pro-inflammatory and way worsen cardiac function, kidney function, diabetes and its complications and may even contribute to obesity.

Bioactive Plant Metabolites in the Management of Non-Communicable Metabolic Diseases: Looking at Opportunities beyond the Horizon.

There has been an unprecedented worldwide rise in non-communicable metabolic diseases (NCDs), particularly cardiovascular diseases (CVD) and diabetes. While modern pharmacotherapy has decreased the mortality in the existing population, it has failed to stem the rise. Furthermore, a large segment of the world population cannot afford expensive pharmacotherapy. Therefore, there is an urgent need for inexpensive preventive measures to control the rise in CVD and diabetes and associated co-morbidities. The purpose of this review is to explore the role of food bioactives in prevention of NCDs. To this end, we have critically analyzed the possible utility of three classes of food bioactives: (a) resistant starch, a metabolically resistant carbohydrate known to favorably modulate insulin secretion and glucose metabolism; (b) cyclo (His-Pro), a food-derived cyclic dipeptides; and (c) polyphenol-rich berries. Finally, we have also briefly outlined the strategies needed to prepare these food-bioactives for human use.

Contribution of dietary advanced glycation end products (AGE) to circulating AGE: role of dietary fat.

The purpose of this pilot study was to determine whether macronutrient content (low-fat v. high-fat diet) influences an indicator of advanced glycation end products (AGE), N(ε) carboxymethyl-lysine (CML), in the context of a 1-d, high-AGE diet. The effect of the diets on inflammatory markers was also assessed. A total of nineteen overweight and obese adults (nine men and ten women) without known disease were recruited to participate in a crossover challenge of a high-fat, high-AGE (HFHA) and low-fat, high-AGE (LFHA) diet. In each phase patients had fasting blood drawn, followed by consumption of a high-fat or low-fat breakfast test meal, then three postprandial blood draws at 1, 2 and 3 h after consuming the test meal. After consuming high-AGE meals for the remainder of the day, participants returned the next day for a follow-up analysis. A different pattern in the 3-h post-meal CML and soluble receptor for AGE response to the two diets was observed (P=0·01 and 0·05, respectively). No change in serum CML was observed following consumption of a LFHA breakfast (535 (25th-75th percentile 451-790) to 495 (25th-75th percentile 391-682) ng/ml; P=0·36), whereas a rise in CML occurred after the HFHA breakfast (463 (25th-75th percentile 428-664) to 578 (25th-75th percentile 474-865) ng/ml; P=0·05). High sensitivity C-reactive protein and high molecular weight adiponectin were not affected by either diet. These findings suggest that dietary CML may not be as important in influencing serum CML as other dietary factors. In addition, acute exposure to dietary CML may not influence inflammation in adults without diabetes or kidney disease. This is contrary to previous findings.

Serum soluble receptor for advanced glycation end products correlates inversely with measures of adiposity in young adults.

Advanced glycation end products (AGEs) may promote inflammation by interacting with the receptor for advanced glycation end products. Serum soluble receptor for advanced glycation end products (sRAGE), a form of receptor for advanced glycation end products thought to mediate AGE's inflammatory properties, is decreased in diabetes mellitus and coronary artery disease. Evidence in older adults suggests that sRAGE is depressed in individuals without current disease who are obese; however, 2 studies have failed to find this correlation. We hypothesized that sRAGE would be inversely correlated with adiposity and positively correlated with inflammation, even in apparently healthy, young adults. By considering adults of body mass index (BMI) varying from normal weight to overweight and obese, we aimed to define how closely AGEs and sRAGE correlate with adiposity and other indicators of metabolic stress. Anthropometric measurements and fasting blood samples were obtained from participants (n = 69). Sera were analyzed for sRAGE, n-epsilon carboxy-methyl-lysine, a measure of AGEs, and high sensitivity C-reactive protein. High molecular weight adiponectin, glucose, insulin, total cholesterol, high-density lipoprotein, and triacylglycerol were also assessed (n = 32). Spearman rank correlations were used to evaluate the relationship among indicators of adiposity and biochemical indicators of metabolic health and inflammation. Factors inversely correlated with sRAGE include weight (Rs = -0.397; P = .001), waist circumference (-0.291; P = .015), and BMI (-0.3338; P = .004). High molecular weight adiponectin was positively correlated with sRAGE, and predictors of sRAGE included BMI and total cholesterol. This is the first time these associations have been found in a diverse population of young adults.

Lifestyle and Advanced Glycation End Products (AGEs) Burden: Its Relevance to Healthy Aging.

Uncontrolled continued exposure to oxidative stress is a precursor to many chronic diseases including cancer, diabetes, degenerative disorders and cardiovascular diseases. Of the many known mediators of oxidative stress, reactive oxygen species (ROS) and advanced glycation end products (AGEs) are the most studied. In the present review, we have summarized current data on the origin of circulating AGEs, discussed issues associated with reliable assessment of its steady state level, and changes in its level with age and select metabolic diseases. Lastly, we have made recommendations about life style changes that may decrease AGEs burden to promote healthy aging.

Prescribing personalized nutrition for cardiovascular health: are we ready?

Of all chronic metabolic diseases, cardiovascular disease (CVD) is the leading cause of death worldwide. Most research over the past 100 years show a link between CVD and lifestyle, including diet; thus, public health messages have focused on modifications of the diet to better manage this disease. Despite this effort, the CVD mortality rate continues to rise. Therefore, is it possible that this failure may be due to individual variability in response to dietary recommendations? The elucidation of the structure of the human genome combined with the knowledge that nutrients are capable of modifying gene expression and genetic variability regulates how individuals respond to a diet have led to the possibility of personalized nutrition for disease prevention. While this possibility is real for the future, our current understanding of nutrient-gene interactions for CVD is limited, making personalized nutrition therapy difficult at this time. With advances in nutritional genomics, in the near future, dietitians and nutritionists will be able to give personalized nutritional advice based on a combination of lifestyle factors and genetics.

Measurement and validation of the nature of salivary adiponectin.

Adiponectin (Ad) is an adipocyte-derived hormone that plays an essential role in regulating insulin sensitivity, inflammation, and atherogenesis. Levels of some hormones in saliva change in a fashion similar to that in plasma in response to a disease or physiological condition. Since saliva is an easy to obtain biological fluid, measurements of salivary hormonal changes are preferred in diagnoses and treatments. Therefore, it was of interest to examine the nature of salivary Ad. While there have been two publications in the literature reporting presence of Ad in human saliva, the nature of salivary Ad has not been characterized. To this end, we investigated the effect of sample dilution on the measurement of Ad in saliva. To our surprise, we observed an increase in measurable level of Ad in saliva on sample dilution. One explanation for this paradoxical observation may be the presence of inhibitor(s) of Ad/anti-Ad binding in saliva that following dilution relieves the inhibitory effect. Working with this hypothesis, we were able to demonstrate the presence of an inhibitor in saliva that co-eluted with the dimeric form of Ad and was capable of inhibiting Ad assay. The presence of such inhibitor(s) may lead to underestimation of Ad in saliva.