Liza Valentin-Blasini

Urinary perchlorate and thyroid hormone levels in adolescent and adult men and women living in the United States.

Background Perchlorate is commonly found in the environment and known to inhibit thyroid function at high doses. Assessing the potential effect of low-level exposure to perchlorate on thyroid function is an area of ongoing research. Objectives We evaluated the potential relationship between urinary levels of perchlorate and serum levels of thyroid stimulating hormone (TSH) and total thyroxine (T4) in 2,299 men and women, ≥ 12 years of age, participating in the National Health and Nutrition Examination Survey (NHANES) during 2001–2002. Methods We used multiple regression models of T4 and TSH that included perchlorate and covariates known to be or likely to be associated with T4 or TSH levels: age, race/ethnicity, body mass index, estrogen use, menopausal status, pregnancy status, premenarche status, serum C-reactive protein, serum albumin, serum cotinine, hours of fasting, urinary thiocyanate, urinary nitrate, and selected medication groups. Results Perchlorate was not a significant predictor of T4 or TSH levels in men. For women overall, perchlorate was a significant predictor of both T4 and TSH. For women with urinary iodine < 100 μg/L, perchlorate was a significant negative predictor of T4 (p < 0.0001) and a positive predictor of TSH (p = 0.001). For women with urinary iodine ≥ 100 μg/L, perchlorate was a significant positive predictor of TSH (p = 0.025) but not T4 (p = 0.550). Conclusions These associations of perchlorate with T4 and TSH are coherent in direction and independent of other variables known to affect thyroid function, but are present at perchlorate exposure levels that were unanticipated based on previous studies.

Perchlorate Exposure of the US Population, 2001-2002

Perchlorate is commonly found in the environment and can impair thyroid function at pharmacological doses. As a result of the potential for widespread human exposure to this biologically active chemical, we assessed perchlorate exposure in a nationally representative population of 2820 US residents, ages 6 years and older, during 2001 and 2002 as part of the National Health and Nutrition Examination Survey (NHANES). We found detectable levels of perchlorate (>0.05 μg/l) in all 2820 urine samples tested, indicating widespread human exposure to perchlorate. Urinary perchlorate levels were distributed in a log normal fashion with a median of 3.6 μg/l (3.38 μg/g creatinine) and a 95th percentile of 14 μg/l (12.7 μg/g creatinine). When geometric means of urinary perchlorate levels were adjusted for age, fasting, sex and race-ethnicity, we found significantly higher levels of urinary perchlorate in children compared with adolescents and adults. We estimated total daily perchlorate dose for each adult (ages 20 years and older), based on urinary perchlorate, urinary creatinine concentration and physiological parameters predictive of creatinine excretion rate. The 95th percentile of the distribution of estimated daily perchlorate doses in the adult population was 0.234 μg/kg-day [CI 0.202–0.268 μg/kg-day] and is below the EPA reference dose (0.7 μg/kg-day), a dose estimated to be without appreciable risk of adverse effects during a lifetime of exposure. These data provide the first population-based assessment of the magnitude and prevalence of perchlorate exposure in the US.

Thyroid-stimulating hormone increases active transport of perchlorate into thyroid cells

Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct measurement techniques. To demonstrate intracellular perchlorate accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1-10 microM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 microM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 microM) did not inhibit cell proliferation after 72 h of incubation. In conclusion, perchlorate is actively transported into thyroid cells and does not inhibit cell proliferation.

HPLC-MS/MS method for the measurement of seven phytoestrogens in human serum and urine

The elevated exposure of children to hormonally active dietary phytoestrogens has led to the need for rapid, sensitive, and precise assays for phytoestrogen metabolites in physiological matrices. Here we report the development of a high-performance liquid chromatography (HPLC) MS/MS method for the quantitative detection of seven phytoestrogens in human serum and urine. The method uses enzymatic deconjugation of the phytoestrogen metabolites followed by solid phase extraction (SPE) and reverse-phase HPLC. The phytoestrogens are detected using a Sciex API III heated nebulizer atmospheric pressure chemical ionization (HN-APCI) interface coupled with tandem mass spectrometry. This method allows the detection of the primary dietary phytoestrogens (isoflavones and lignans) in human serum and urine with limits of detection (LODs) in the low parts per billion range. The combination of tandem mass spectrometry and chromatographic separation of the analytes helps ensure the selectivity of the method. Stable isotope-labeled internal standards for all seven analytes improve the precision of the assay, resulting in interday CV values of <10% for most compounds studied. The accuracy and precision of the method were monitored over time using quality control (QC) samples containing known amounts of phytoestrogens. The majority of phytoestrogens in human sera and urine are present as their glucuronide and sulfate conjugates. Therefore, the thoroughness of deconjugation for each sample was monitored by the addition of a conjugated internal standard and subsequent detection of deconjugated compound. This method proves to be efficacious for measuring baseline urinary phytoestrogen levels in the American population and should prove useful for assessing the modulatory effects of dietary phytoestrogens on endocrine disrupter action in children.

Simultaneous determination of six mercapturic acid metabolites of volatile organic compounds in human urine.

The widespread exposure to potentially harmful volatile organic compounds (VOCs) merits the development of practical and accurate exposure assessment methods. Measuring the urinary concentrations of VOC mercapturic acid (MA) metabolites provides noninvasive and selective information about recent exposure to certain VOCs. We developed a liquid chromatography-tandem mass spectrometry method for quantifying urinary levels of six MAs: N-acetyl-S-(2-carboxyethyl)-L-cysteine (CEMA), N-acetyl-S-(3-hydroxypropyl)-L-cysteine (HPMA), N-acetyl-S-(2-hydroxy-3-butenyl)-L-cysteine (MHBMA), N-acetyl-S-(3,4-dihydroxybutyl)-L-cysteine (DHBMA), N-acetyl-S-(2-hydroxyethyl)-L-cysteine (HEMA), and N-acetyl-S-(phenyl)-L-cysteine (PMA). The method provides good accuracy (102% mean accuracy) and high precision (3.5% mean precision). The sensitivity (limits of detection of 0.01-0.20 microg/L) and wide dynamic detection range (0.025-500 microg/L) make this method suitable for assessing VOC exposure of minimally exposed populations and those with significant exposures, such as cigarette smokers. We used this method to quantify MA levels in urine collected from smokers and nonsmokers. Median levels of creatinine-corrected CEMA, HPMA, MHBMA, DHBMA, HEMA, and PMA among nonsmokers (n = 59) were 38.1, 24.3, 21.3, 104.7, 0.9, and 0.5 microg/g creatinine, respectively. Among smokers (n = 61), median levels of CEMA, HPMA, MHBMA, DHBMA, HEMA, and PMA were 214.4, 839.7, 10.2, 509.7, 2.2, and 0.9 microg/g creatinine, respectively. All VOC MAs measured were higher among smokers than among nonsmokers, with the exception of MHBMA.

Urinary and serum concentrations of seven phytoestrogens in a human reference population subset

Diets rich in naturally occurring plant estrogens (phytoestrogens) are strongly associated with a decreased risk for cancer and heart disease in humans. Phytoestrogens have estrogenic and, in some cases, antiestrogenic and antiandrogenic properties, and may contribute to the protective effect of some diets. However, little information is available about the levels of these phytoestrogens in the general US population. Therefore, levels of phytoestrogens were determined in urine (N=199) and serum (N=208) samples taken from a nonrepresentative subset of adults who participated in NHANES III, 1988–1994. The phytoestrogens quantified were the lignans (enterolactone, enterodiol, matairesinol); the isoflavones (genistein, daidzein, equol, O-desmethylangolensin); and coumestrol (urine only). Phytoestrogens with the highest mean urinary levels were enterolactone (512 ng/ml), daidzein (317 ng/ml), and genistein (129 ng/ml). In serum, the concentrations were much less and the relative order was reversed, with genistein having the highest mean level (4.7 ng/ml), followed by daidzein (3.9 ng/ml) and enterolactone (3.6 ng/ml). Highly significant correlations of phytoestrogen levels in urine and serum samples from the same persons were observed for enterolactone, enterodiol, genistein, and daidzein. Determination of phytoestrogen concentrations in large study populations will give a better insight into the actual dietary exposure to these biologically active compounds in the US population.

Urinary phytoestrogen concentrations in the U.S. population (1999–2000)

We report population-based urinary concentrations of phytoestrogens stratified by age, sex, and composite racial/ethnic variables. We measured the isoflavones — genistein, daidzein, equol, and O-desmethylangolensin (O-DMA) — and the lignans — enterolactone and enterodiol — in approximately 2500 urine samples from individuals aged 6 years and older who participated in the National Health and Nutrition Examination Survey (NHANES) in 1999 and 2000. We detected all phytoestrogens in over 70% of the samples analyzed; enterolactone was detected in the highest concentrations, and daidzein was detected with the highest frequency. The geometric means for each phytoestrogen were as follows: genistein, 22.3 μg/g; daidzein, 68.6 μg/g; equol, 7.65 μg/g; O-DMA, 3.95 μg/g; enterolactone, 217 μg/g; and enterodiol, 24.3 μg/g creatinine. The 95th percentiles for each phytoestrogen were as follows: genistein, 380 μg/g; daidzein, 944 μg/g; equol, 50.3 μg/g; O-DMA, 217 μg/g; enterolactone, 2240 μg/g; and enterodiol, 240 μg/g creatinine. Multivariate analyses showed statistically significant differences among many of the demographic subgroups. Adolescents had higher concentrations of genistein and equol than adults. Non-Hispanic whites had higher concentrations of enterodiol and equol than Mexican Americans or non-Hispanic blacks. Non-Hispanic whites also had higher concentrations of enterolactone and O-DMA than Mexican Americans. Mexican Americans had higher concentrations of genistein than non-Hispanic blacks; however, the opposite was found for O-DMA. Determination of phytoestrogen exposure in the US population will help us to better understand phytoestrogen consumption in the US and will assist us in elucidating the potential role of phytoestrogens in protecting against cancer and heart disease.

Analysis of Perchlorate in Dried Blood Spots Using Ion Chromatography and Tandem Mass Spectrometry

Because of health concerns surrounding in utero and neonatal exposure to perchlorate, we developed a method for analyzing perchlorate in the dried blood spots (DBS) of newborns. Ion chromatography was interfaced with electrospray ionization tandem mass spectrometry to measure blood perchlorate levels in DBS samples as low as 0.10 ng/mL. Measurement of perchlorate in DBS indicated good precision (relative standard deviations ranging from 5.8% to 16.2%) and accuracy (% difference values ranging from -11.3% to -12.1%). Perchlorate was detectable in 100% of the DBS collected from 100 newborns. These samples had a median blank-adjusted concentration of 1.88 ng/mL. Such data support the utility of this method to quantify perchlorate levels in DBS samples. Applying this method to analyze neonatal DBS will improve perchlorate exposure assessments of this susceptible population.

Goitrogenic anions, thyroid-stimulating hormone, and thyroid hormone in infants.

Background Environmental exposure of infants to perchlorate, thiocyanate, nitrate, might interfere with thyroid function. U.S. women with higher background perchlorate exposure have higher thyroid-stimulating hormone (TSH) and lower thyroxine (T4). There are no studies with individual measures of thyroid function and these goitrogens available in infants. Objective We examined the association of urinary perchlorate, nitrate, iodide, and thiocyanate with urinary T4 and TSH in infants and whether that association differed by sex or iodide status. Methods We used data and samples from the Study of Estrogen Activity and Development, which assessed hormone levels of full-term infants over the first 12 months of life. The study included 92 full-term infants between birth and 1 year of age seen up to four times. Perchlorate, thiocyanate, nitrate, and iodide were measured in 206 urine samples; TSH and T4 and were measured in urines and in 50 blood samples. Results In separate mixed models, adjusting for creatinine, age, sex, and body mass index, infants with higher urinary perchlorate, nitrate or thiocyanate had higher urinary TSH. With all three modeled, children with higher nitrate and thiocyanate had higher TSH, but higher perchlorate was associated with TSH only in children with low iodide. Unexpectedly, exposure to the three chemicals was generally associated with higher T4. Conclusions The association of perchlorate exposure with increased urinary TSH in infants with low urinary iodide is consistent with previous findings. Higher thiocyanate and nitrate exposure were also associated with higher TSH in infants.

Perchlorate in the Feed-Dairy Continuum of the Southwestern United States

Perchlorate has the potential to cause thyroid dysfunction by inhibiting iodide uptake by the sodium iodide symporter. Perchlorate-contaminated waters may lead to human exposure through drinking water and food chain transfer in crops by way of irrigation water. Perchlorate has been found in dairy milk collected nationally and internationally. This study was conducted to evaluate perchlorate in the feed-dairy continuum in the southwestern United States. All feed products collected at dairies in this study had detectable levels of perchlorate as analyzed by ion chromatography-tandem mass spectrometry. The calculated total perchlorate intake across dairies ranged from 1.9 to 12.7 mg/cow per day. The variation in total perchlorate intake across dairies was largely associated with variation in forage and silage products. Alfalfa products were the single most important source of perchlorate intake variability among dairies. The estimated perchlorate intake from drinking water ranged from 0.01 mg per cow per day and was generally less than 2% of the total perchlorate intake. The perchlorate content of milk ranged from 0.9 to 10.3 microg/L and was similar to levels reported by the Food and Drug Administrations Total Diet Study. The perchlorate content of milk was significantly related to the presence of perchlorate in feed but the variation of perchlorate in milk could not be explained by feed intake alone.