Urinary excretion of sodium, potassium, and chloride, but not iodine, varies by timing of collection in a 24-hour calibration study.

Because of the logistic complexity, excessive respondent burden, and high cost of conducting 24-h urine collections in a national survey, alternative strategies to monitor sodium intake at the population level need to be evaluated. We conducted a calibration study to assess the ability to characterize sodium intake from timed-spot urine samples calibrated to a 24-h urine collection. In this report, we described the overall design and basic results of the study. Adults aged 18-39 y were recruited to collect urine for a 24-h period, placing each void in a separate container. Four timed-spot specimens (morning, afternoon, evening, and overnight) and the 24-h collection were analyzed for sodium, potassium, chloride, creatinine, and iodine. Of 481 eligible persons, 407 (54% female, 48% black) completed a 24-h urine collection. A subsample (n = 133) collected a second 24-h urine 4-11 d later. Mean sodium excretion was 3.54 ± 1.51 g/d for males and 3.09 ± 1.26 g/d for females. Sensitivity analysis excluding those who did not meet the expected creatinine excretion criterion showed the same results. Day-to-day variability for sodium, potassium, chloride, and iodine was observed among those collecting two 24-h urine samples (CV = 16-29% for 24-h urine samples and 21-41% for timed-spot specimens). Among all race-gender groups, overnight specimens had larger volumes (P < 0.01) and lower sodium (P < 0.01 to P = 0.26), potassium (P < 0.01), and chloride (P < 0.01) concentrations compared with other timed-spot urine samples, although the differences were not always significant. Urine creatinine and iodine concentrations did not differ by the timing of collection. The observed day-to-day and diurnal variations in sodium excretion illustrate the importance of accounting for these factors when developing calibration equations from this study.

Summary of an NIH workshop to identify research needs to improve the monitoring of iodine status in the United States and to inform the DRI.

The Office of Dietary Supplements (ODS) at the NIH sponsored a workshop on May 12-13, 2011, to bring together representatives from various NIH institutes and centers as a first step in developing an NIH iodine research initiative. The workshop also provided an opportunity to identify research needs that would inform the dietary reference intakes for iodine, which were last revised in 2001. Iodine is required throughout the life cycle, but pregnant women and infants are the populations most at risk of deficiency, because iodine is required for normal brain development and growth. The CDC monitors iodine status of the population on a regular basis, but the status of the most vulnerable populations remains uncertain. The NIH funds very little investigator-initiated research relevant to iodine and human nutrition, but the ODS has worked for several years with a number of other U.S. government agencies to develop many of the resources needed to conduct iodine research of high quality (e.g., validated analytical methods and reference materials for multiple types of samples). Iodine experts, scientists from several U.S. government agencies, and NIH representatives met for 2 d to identify iodine research needs appropriate to the NIH mission.

Breast cancer risk among women under 55 years of age by joint effects of usage of oral contraceptives and hormone replacement therapy.

OBJECTIVE: To assess effects on breast cancer risk of exposure to both oral contraceptives and menopausal hormones, an increasingly common exposure. DESIGN: A case-control study of breast cancer among women under the age of 55 years in Atlanta, GA involving 1,031 cases and 919 population controls was conducted. RESULTS: Ever use of oral contraceptives was associated with a relative risk of 1.1 (95% 0.9-1.4), whereas the relative risk for hormone replacement therapy was 0.9 (95% CI 0.7-1.2). Seventeen percent of the cases versus 19% of the population controls reported exposure to both agents, resulting in a relative risk of 1.0 (95% CI 0.7-1.4) relative to those unexposed to either preparation. Although there was little variation in risk associated with joint effects by either age or race, there were statistically nonsignificant elevations in risk for this exposure among women who had experienced a natural menopause (relative risk = 2.0, 95% CI 0.7-5.6), were relatively thin (relative risk = 1.5, 0.8-3.0), or who had a first degree relative with breast cancer (relative risk = 2.0, 0.6-7.0). When joint effects of longer term use of both agents were considered, subjects who reported use of oral contraceptives for 10 or more years and hormone replacement for 3 or more years had a relative risk of 3.2 (95% CI 1.4-7.4) compared with nonusers of either preparation. CONCLUSIONS: Although our results must be cautiously interpreted given small numbers within subgroups, they raise concern and emphasize the need for further evaluation on breast cancer risk of the increasingly common exposure to both oral contraceptives and hormone replacement therapy.

General and abdominal obesity and survival among young women with breast cancer.

Among postmenopausal women, obesity is linked to increased risk of breast cancer and poorer subsequent survival. For premenopausal women, obesity may reduce incidence, but less is known about its effect on prognosis, particularly for abdominal obesity. This study investigated whether general or abdominal obesity at diagnosis influenced survival in a cohort of young women with breast cancer. A population-based follow-up study was conducted among 1,254 women ages 20 to 54 who were diagnosed with invasive breast cancer between 1990 and 1992 in Atlanta or New Jersey. Women were interviewed within several months of diagnosis and asked about their weight and height at age 20 and in the year before diagnosis. Study personnel did anthropometric measures at the interview. With 8 to 10 years of follow-up, all-cause mortality status was determined using the National Death Index (n = 290 deaths). Increased mortality was observed for women who were obese [body mass index (BMI), > or =30] at the time of interview compared with women of ideal weight [BMI, 18.5-24.9; stage- and income-adjusted hazard ratio (HR), 1.48; 95% confidence interval (95% CI), 1.09-2.01]. A similar result was seen for the highest versus lowest quartile of waist-to-hip ratio (HR, 1.52; 95% CI, 1.05-2.19). Strong associations with mortality were found for women who were obese at age 20 (HR, 2.49; 95% CI, 1.15-5.37) or who were overweight/obese (BMI, > or =25) at both age 20 and the time of interview (HR, 2.22; 95% CI, 1.45-3.40). This study provides evidence that breast cancer survival is reduced among younger women with general or abdominal obesity.

Research needs for assessing iodine intake, iodine status, and the effects of maternal iodine supplementation.

The Office of Dietary Supplements of the NIH convened 3 workshops on iodine nutrition in Rockville, Maryland, in 2014. The purpose of the current article is to summarize and briefly discuss a list of research and resource needs developed with the input of workshop participants. This list is composed of the basic, clinical, translational, and population studies required for characterizing the benefits and risks of iodine supplementation, along with related data, analyses, evaluations, methods development, and supporting activities. Ancillary studies designed to use the participant, biological sample, and data resources of ongoing and completed studies (including those not originally concerned with iodine) may provide an efficient, cost-effective means to address some of these research and resource needs. In the United States, the foremost question is whether neurobehavioral development in the offspring of mildly to moderately iodine-deficient women is improved by maternal iodine supplementation during pregnancy. It is important to identify the benefits and risks of iodine supplementation in all population subgroups so that supplementation can be targeted, if necessary, to avoid increasing the risk of thyroid dysfunction and related adverse health effects in those with high iodine intakes. Ultimately, there will be a need for well-designed trials and other studies to assess the impact of maternal supplementation on neurodevelopmental outcomes in the offspring. However, 2 basic information gaps loom ahead of such a study: the development of robust, valid, and convenient biomarkers of individual iodine status and the identification of infant and toddler neurobehavioral development endpoints that are sensitive to mild maternal iodine deficiency during pregnancy and its reversal by supplementation.

Assessing iodine intake, iodine status, and the effects of maternal iodine supplementation: introduction to articles arising from 3 workshops held by the NIH Office of Dietary Supplements.

The NIH Office of Dietary Supplements (ODS) convened 3 workshops on iodine nutrition in 2014, each held in Rockville, Maryland. These workshops were part of the ongoing ODS Iodine Initiative, begun in 2011 in response to concerns that US pregnant women may be at risk of iodine deficiency and that a high fraction of prenatal dietary supplements do not contain the recommended amounts of iodine. The primary purpose of the workshops was to consider the data and resources necessary to evaluate the clinical and public health benefits and risks of maternal iodine supplementation in the United States. The first workshop focused on the assessment of iodine intake, the second focused on the assessment of iodine status, and the third focused on the design and interpretation of clinical trials of maternal iodine supplementation. Here we provide the background of the ODS Iodine Initiative, summarize the 3 workshops held in 2014, and introduce the articles that arose from the workshops and are published in this supplement issue.

Iodine in food- and dietary supplement-composition databases.

The US Food and Drug Administration (FDA) and the Nutrient Data Laboratory (NDL) of the USDA Agricultural Research Service have worked independently on determining the iodine content of foods and dietary supplements and are now harmonizing their efforts. The objective of the current article is to describe the harmonization plan and the results of initial iodine analyses accomplished under that plan. For many years, the FDA's Total Diet Study (TDS) has measured iodine concentrations in selected foods collected in 4 regions of the country each year. For more than a decade, the NDL has collected and analyzed foods as part of the National Food and Nutrient Analysis Program; iodine analysis is now being added to the program. The NDL recently qualified a commercial laboratory to conduct iodine analysis of foods by an inductively coupled plasma mass spectrometry (ICP-MS) method. Co-analysis of a set of samples by the commercial laboratory using the ICP-MS method and by the FDA laboratory using its standard colorimetric method yielded comparable results. The FDA recently reviewed historical TDS data for trends in the iodine content of selected foods, and the NDL analyzed samples of a limited subset of those foods for iodine. The FDA and the NDL are working to combine their data on iodine in foods and to produce an online database that can be used for estimating iodine intake from foods in the US population. In addition, the NDL continues to analyze dietary supplements for iodine and, in collaboration with the NIH Office of Dietary Supplements, to publish the data online in the Dietary Supplement Ingredient Database. The goal is to provide, through these 2 harmonized databases and the continuing TDS focus on iodine, improved tools for estimating iodine intake in population studies.

Comparison of 2 methods for estimating the prevalences of inadequate and excessive iodine intakes.

BACKGROUND: Prevalences of iodine inadequacy and excess are usually evaluated by comparing the population distribution of urinary iodine concentration (UIC) in spot samples with established UIC cutoffs. To our knowledge, until now, dietary intake data have not been assessed for this purpose. OBJECTIVE: Our objective was to compare 2 methods for evaluating the prevalence of iodine inadequacy and excess in sex- and life stage-specific subgroups of the US population: one that uses UIC cutoffs, and one that uses iodine intake cutoffs. DESIGN: By using the iodine concentrations of foods measured in the US Food and Drug Administration's Total Diet Study (TDS), dietary intake data from the NHANES 2003-2010, and a file that maps each NHANES food to a TDS food with similar ingredients, we estimated each NHANES participant's iodine intake from each NHANES food as the mean iodine concentration of the corresponding TDS food in samples gathered over the same 2-y period. We calculated prevalences of iodine inadequacy and excess in each sex- and life stage-specific subgroup by both the UIC cutoff method and the iodine intake cutoff method-using the UIC values and dietary intakes reported for NHANES participants who provided both types of data-and compared the prevalences across methods. RESULTS: We found lower prevalences of iodine inadequacy across all sex- and life stage-specific subgroups with the iodine intake cutoff method than with the UIC cutoff method; for pregnant females, the respective prevalences were 5.0% and 37.9%. For children aged ≤8 y, the prevalence of excessive iodine intake was high by either method. CONCLUSIONS: The consideration of dietary iodine intake from all sources may provide a more complete understanding of population prevalences of iodine inadequacy and excess and thus better inform dietary guidance than consideration of UIC alone. Methods of adjusting UIC for within-person variation are needed to improve the accuracy of prevalence assessments based on UIC.

Suggested guidelines for articles about botanical dietary supplements.

Recently, The American Journal of Clinical Nutrition (AJCN) began reviewing articles about dietary supplements. The purpose of this commentary is to provide guidelines to authors and reviewers for articles on one category of supplement ingredients, botanicals. The botanicals in the studies published by the AJCN tend to fall into 1 of 2 groups: 1) plants as foods containing nonessential bioactive constituents that may provide health benefits beyond basic nutrition, and 2) plants as herbs, specifically those used as phytomedicines. Research in these areas is relevant to clinical nutrition, but both topics represent relatively new territory to many AJCN reviewers, readers, and contributors. Although studies of botanicals are unique in many respects, the research should be evaluated with the same basic criteria applied to other types of investigations. For example, a study cannot be evaluated or replicated unless the test materials are properly identified and characterized. Investigators must provide an accurate and complete description of the botanical test material regardless of whether it is a finished product, commercial ingredient, extract, or single chemical constituent. For herbal preparations, investigators are advised to follow the criteria used by researchers in the field of pharmacognosy. Finally, the quality of research related to botanical dietary supplements would be improved and cross-study comparisons facilitated if standard reference materials and certified methods of analysis were more broadly available.

Iron intake and regulation: implications for iron deficiency and iron overload.

Although iron deficiency anemia is the most common nutritional deficiency worldwide and in the United States, the health effects of iron overload merit increased attention. In the United States, public health interventions such as fortification and enrichment of foods with iron were undertaken to reduce the prevalence of iron deficiency anemia and improve health. These measures, along with iron supplementation, remain controversial, because additional exposure to dietary iron places some segments of the population at increased risk of iron excess. The health consequences of unmistakable iron excess are exemplified by hemochromatosis, an iron storage disease associated with liver damage further exacerbated by alcohol consumption. Progressive liver damage associated with this condition is generally attributed to increased oxidative stress. In otherwise healthy individuals, more modest levels of iron storage may occur if iron is provided by supplements or otherwise added to the food supply. Increased iron intake and storage have been linked to a variety of chronic diseases. The associations are not firmly established but are of considerable public health importance.

Comparison of population iodine estimates from 24-hour urine and timed-spot urine samples.

BACKGROUND: Median urine iodine concentration (UIC; µg/L) in spot urine samples is recommended for monitoring population iodine status. Other common measures are iodine:creatinine ratio (I/Cr; µg/g) and estimated 24-hour urine iodine excretion (UIE; I/Cr × predicted 24-hour Cr; µg/day). Despite different units, these measures are often used interchangeably, and it is unclear how they compare with the reference standard 24-hour UIE. METHODS: Volunteers aged 18-39 years collected all their urine samples for 24 hours (n=400). Voids from morning, afternoon, evening, overnight, and a composite 24-hour sample were analyzed for iodine. We calculated median observed 24-hour UIE and 24-hour UIC, and spot UIC, I/Cr, and two measures of estimated UIE calculated using predicted 24-hour Cr from published estimates by Kesteloot and Joosens (varies by age and sex) and published equations by Mage et al. (varies by age, sex, race, and anthropometric measures). We examined mean differences and relative difference across iodine excretion levels using Bland-Altman plots. RESULTS: Median 24-hour UIE was 173.6 µg/day and 24-hour UIC was 144.8 µg/L. From timed-spot urine samples, estimates were: UIC 147.3-156.2 µg/L; I/Cr 103.6-114.3 µg/g, estimated 24-hour UIE (Kesteloot and Joosens) 145.7-163.3 µg/day; and estimated 24-hour UIE (Mage) 176.5-187.7 µg/day. Iodine measures did not vary consistently by timing of spot urine collection. Compared with observed 24-hour UIE, on average, estimated (Mage) 24-hour UIE was not significantly different, while estimated 24-hour UIE (Kesteloot and Joosens) was significantly different for some ethnicity/sex groups. Compared with 24-hour UIC, on average, spot UIC did not differ. CONCLUSIONS: Estimates of UIC, I/Cr, and estimated 24-hour UIE (I/Cr × predicted 24-hour Cr) from spot urine samples should not be used interchangeably. Estimated 24-hour UIE, where predicted 24-hour Cr varies by age, sex, ethnicity, and anthropometric measures and was calculated with prediction equations using data from the sample, was more comparable to observed 24-hour UIE than when predicted 24-hour Cr was from published estimates from a different population. However, currently no cutoffs exist to interpret population estimated 24-hour UIE values.

Iodine insufficiency: a global health problem?

As a result of collaborative efforts with international organizations and the salt industry, many developing and developed countries practice universal salt iodization (USI) or have mandatory salt fortification programs. As a consequence, the prevalence of iodine deficiency decreased dramatically. The United States and Canada are among the few developed countries that do not practice USI. Such an undertaking would require evidence of deficiency among vulnerable population groups, including pregnant women, newborns, and developing infants. Government agencies in the United States rely heavily on data from NHANES to assess the iodine status of the general population and pregnant women in particular. NHANES data suggest that pregnant women in the United States remain mildly deficient. This is important, because the developing fetus is dependent on maternal iodine intake for normal brain development throughout pregnancy. Professional societies have recommended that pregnant and lactating women, or those considering pregnancy, consume a supplement providing 150 µg iodine daily. The United States and Canada collaborate on the daily recommended intake and are also confronted with the challenge of identifying the studies needed to determine if USI is likely to be beneficial to vulnerable population groups without exposing them to harm.

Validity of predictive equations for 24-h urinary sodium excretion in adults aged 18-39 y.

BACKGROUND: Collecting a 24-h urine sample is recommended for monitoring the mean population sodium intake, but implementation can be difficult. OBJECTIVE: The objective was to assess the validity of published equations by using spot urinary sodium concentrations to predict 24-h sodium excretion. DESIGN: This was a cross-sectional study, conducted from June to August 2011 in metropolitan Washington, DC, of 407 adults aged 18-39 y, 48% black, who collected each urine void in a separate container for 24 h. Four timed voids (morning, afternoon, evening, and overnight) were selected from each 24-h collection. Published equations were used to predict 24-h sodium excretion with spot urine by specimen timing and race-sex subgroups. We examined mean differences with measured 24-h sodium excretion (bias) and individual differences with the use of Bland-Altman plots. RESULTS: Across equations and specimens, mean bias in predicting 24-h sodium excretion for all participants ranged from -267 to 1300 mg (Kawasaki equation). Bias was least with International Cooperative Study on Salt, Other Factors, and Blood Pressure (INTERSALT) equations with morning (-165 mg; 95% CI: -295, 36 mg), afternoon (-90 mg; -208, 28 mg), and evening (-120 mg; -230, -11 mg) specimens. With overnight specimens, mean bias was least when the Tanaka (-23 mg; 95% CI: -141, 95 mg) or Mage (-145 mg; -314, 25 mg) equations were used but was statistically significant when using the Tanaka equations among females (216 to 243 mg) and the Mage equations among races other than black (-554 to -372 mg). Significant over- and underprediction occurred across individual sodium excretion concentrations. CONCLUSIONS: Using a single spot urine, INTERSALT equations may provide the least biased information about population mean sodium intakes among young US adults. None of the equations evaluated provided unbiased estimates of individual 24-h sodium excretion.

Computer access to research on dietary supplements: a database of federally funded dietary supplement research.

Dietary supplement use is prevalent in the United States, but support for supplement research has been relatively modest and only recently emphasized at the NIH. The Dietary Supplement Health and Education Act of 1994 led to the creation of the Office of Dietary Supplements (ODS) at the NIH to promote research on dietary supplements. In order to track federally funded dietary supplement research, the ODS developed a database known as Computer Access to Research on Dietary Supplements (CARDS). This article provides an overview of the development and potential uses of the CARDS database. In addition, we report that NIH-funded dietary supplement research steadily increased from fiscal year (FY) 1999 through 2002. The majority of NIH institutes or centers (ICs) funded research relevant to dietary supplements during this time, led by the National Cancer Institute and one of the newest NIH ICs, the National Center for Complementary and Alternative Medicine. CARDS data indicate that NIH-funded dietary supplement research from FY 1999 through 2002 involved primarily vitamins, minerals, botanicals and phytochemicals. Cancer and cardiovascular disease, two of the leading causes of morbidity and mortality in the United States, collectively accounted for almost 45% of the research related to dietary supplements. A variety of types of research studies were funded, with the majority consisting of human intervention studies. This information is useful to evaluate trends in federally funded dietary supplement research, identify research gaps, and help research scientists identify potential sources of NIH funding.

Dietary carotenoids, vegetables, and lung cancer risk in women: the Missouri women's health study (United States).

OBJECTIVE: To examine the effect of specific dietary carotenoids and their primary plant food sources on lung cancer risk in a population-based case-control study of women. METHODS: Data were available for 587 incident primary lung cancer cases and 624 controls frequency matched to cases based on age. A modified version of the 100-item NCI-Block food-frequency questionnaire was used to obtain information concerning usual diet 2-3 years prior to interview. RESULTS: In models adjusted for age, total calorie intake, pack-years of smoking, and education, beta-carotene, beta-cryptoxanthin, lutein + zeaxanthin, and total carotenoid intake were each associated with a significantly lower risk of lung cancer. Several vegetable groups were predictive of lower lung cancer risk, particularly the frequency of total vegetable intake. Individual and total carotenoids were no longer significantly associated with lower lung cancer risk in models adjusted for total vegetable intake. However, total vegetable intake remained significantly inversely associated with risk in models adjusted for total carotenoids. CONCLUSIONS: These results indicate that consumption of a wide variety of vegetables has a greater bearing on lung cancer risk in a population of smoking and nonsmoking women than intake of any specific carotenoid or total carotenoids.

Quantifying epidemiologic risk factors using non-parametric regression: model selection remains the greatest challenge.

Logistic regression is widely used to estimate relative risks (odds ratios) from case-control studies, but when the study exposure is continuous, standard parametric models may not accurately characterize the exposure-response curve. Semi-parametric generalized linear models provide a useful extension. In these models, the exposure of interest is modelled flexibly using a regression spline or a smoothing spline, while other variables are modelled using conventional methods. When coupled with a model-selection procedure based on minimizing a cross-validation score, this approach provides a non-parametric, objective, and reproducible method to characterize the exposure-response curve by one or several models with a favourable bias-variance trade-off. We applied this approach to case-control data to estimate the dose-response relationship between alcohol consumption and risk of oral cancer among African Americans. We did not find a uniquely 'best' model, but results using linear, cubic, and smoothing splines were consistent: there does not appear to be a risk-free threshold for alcohol consumption vis-à-vis the development of oral cancer. This finding was not apparent using a standard step-function model. In our analysis, the cross-validation curve had a global minimum and also a local minimum. In general, the phenomenon of multiple local minima makes it more difficult to interpret the results, and may present a computational roadblock to non-parametric generalized additive models of multiple continuous exposures. Nonetheless, the semi-parametric approach appears to be a practical advance.

Increased risk of early-stage breast cancer related to consumption of sweet foods among women less than age 45 in the United States.

OBJECTIVES: To evaluate the associations of dietary macronutrients, food groups, and eating patterns with risk of breast cancer in a population-based case-control study. METHODS: In this study among women 20-44 years of age, 568 cases with breast cancer and 1451 population-based controls were included. They completed a detailed in-person interview, a self-administered food-frequency questionnaire and were measured for anthropometric indices. Logistic regression was used to estimate odds ratios (OR) and their 95% confidence intervals (CI) of breast cancer, adjusted for age, study site, race, education, alcohol consumption, oral contraceptive usage, smoking status, and body mass index. RESULTS: There was no association between breast cancer risk and intake of calories, macronutrients, or types of fat. Risk of breast cancer was unrelated to intakes of a variety of food groups, including red meats, dairy, high-fat snacks and desserts, or foods high in animal fat. Increased risk was observed for high intake of a food group composed of sweet items, particularly sodas and desserts. Risk increased linearly with percent of calories from sweets and frequency of sweets intake. Consumption of sweets 9.8 or more times per week compared with <2.8 times per week was associated with an adjusted OR of 1.32 (95% CI = 1.0-1.8). This association did not appear to be due to the high-fat foods or carbonated beverages that comprised the food group. Compared with women reporting one or two meals and snacks per day, reduced risks were noted for women reporting six or more (OR = 0.69, 95% CI = 0.4-1.1). CONCLUSIONS: These data suggest a modest relationship between intakes of sweet items with risk of in-situ and localized breast cancer in young women. This relation is consistent with the hypothesized link of high insulin exposure and risk of breast cancer. There was some suggestion that women who ate many times during the day were at reduced risk of disease, which is also consistent with an insulin-related mechanism.

Occupation and breast cancer in women 20-44 years of age (United States).

OBJECTIVE: To examine the relation between breast cancer risk and job history among women 20-44 years of age who participated in a multi-center, population-based, case-control study. METHODS: Participants consisted of women newly diagnosed with breast cancer (1642) and controls identified by random-digit dialing (1494). Details about the three longest jobs were collected and coded by an industrial hygienist. Odds ratios and 95% confidence intervals were calculated and adjusted for age, study site, and other breast cancer risk factors. RESULTS: Several occupational and industrial categories were found to influence breast cancer risk. Stratification of the study population by parity revealed differences in breast cancer risk between the two groups for several occupational categories, including teachers, librarians or counselors (increased risk only among parous women) and natural scientists and mathematicians (decreased risk only among nulliparous women). CONCLUSIONS: This is among the first population-based case-control studies to examine occupational history and breast cancer risk in young women, with the ability to consider a wide array of potential confounders, including reproductive characteristics. This study provides further evidence of an increased breast cancer risk for several occupations and industries. Other findings were not as strongly supported by previous investigations.