A New Interpretation of Relative Importance on an Analysis of Per and Polyfluorinated Alkyl Substances (PFAS) Exposures on Bone Mineral Density.

BACKGROUND: The relative contribution of environmental contaminants is an important, and frequently unanswered, question in human or ecological risk assessments. This interpretation of relative importance allows determination of the overall effect of a set of variables relative to other variables on an adverse health outcome. There are no underlying assumptions of independence of variables. The tool developed and used here is specifically designed for studying the effects of mixtures of chemicals on a particular function of the human body. METHODS: We apply the approach to estimate the contributions of total exposure to six PFAS (perfluorodecanoic acid, perfluorohexane sulfonic acid, 2-(N-methyl-PFOSA) acetate, perfluorononanoic acid, perfluoroundecanoic acid and perfluoroundecanoic acid) to loss of bone mineral density relative to other factors related to risk of osteoporosis and bone fracture, using data from subjects who participated in the US National Health Examination and Nutrition Surveys (NHANES) of 2013-2014. RESULTS: PFAS exposures contribute to bone mineral density changes relative to the following variables: age, weight, height, vitamin D2 and D3, gender, race, sex hormone binding globulin, testosterone, and estradiol. CONCLUSION: We note significant alterations to bone mineral density among more highly exposed adults and significant differences in effects between men and women.

Predicting Exposure to Perfluorinated Alkyl Substances (PFAS) among US Infants.

PFASs have been detected in nearly every serum sample collected over the last two decades from US adults as part of the National Health and Nutrition Examination Survey (NHANES) and are commonly found in other data sets from around the world. However, less is known about infant PFAS exposures, primarily because the collection of infant serum samples is less common and frequently avoided. Cord blood samples are often preferred for chemical exposure assessments because this is thought to provide a good representation of infant serum concentrations, at least at the time of birth. In this paper, we will provide a statistical and probabilistic analysis of what can be expected for infants living in the US using NHANES from 2007 to 2008, which contains a rare subset of infant data. Regulatory efforts that require estimation of exposures among the very youth can be challenging, both because of a lack of data in general and because variability among this most vulnerable population can be uncertain. We report that US infant exposures are extremely common and that serum concentrations remain fairly constant, despite infant growth rates and relatively high caloric and fluid intake, with the possible exception of PFOS. Infant serum PFOS concentrations between months 1 and 3 are consistently higher than at less than one month, even though healthy infants at 1 and 2 months weigh more than they did at birth. This suggests that the babies are exposed to greater concentrations of PFOS after birth or that excretion kinetics differ for this PFAS.

PFAS and Potential Adverse Effects on Bone and Adipose Tissue Through Interactions With PPARγ.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are a widely dispersed, broad class of synthetic chemicals with diverse biological effects, including effects on adipose and bone differentiation. PFAS most commonly occur as mixtures and only rarely, if ever, as single environmental contaminants. This poses significant regulatory questions and a pronounced need for chemical risk assessments, analytical methods, and technological solutions to reduce the risk to public and environmental health. The effects of PFAS on biological systems may be complex. Each may have several molecular targets initiating multiple biochemical events leading to a number of different adverse outcomes. An exposure to mixtures or coexposures of PFAS complicates the picture further. This review illustrates how PFAS target peroxisome proliferator-activated receptors. Additionally, we describe how such activation leads to changes in cell differentiation and bone development that contributes to metabolic disorder and bone weakness. This discussion sheds light on the importance of seemingly modest outcomes observed in test animals and highlights why the most sensitive end points identified in some chemical risk assessments are significant from a public health perspective.

Evaluation of Fetal Exposures to Metals and Metalloids through Meconium Analyses: A Review.

This paper surveys the existing scientific literature on metals concentrations in meconium. We examine some 32 papers that analyzed meconium for aluminum, arsenic, barium, calcium, chromium, copper, iron, lithium, magnesium, manganese, zinc, lead, mercury, manganese, molybdenum, nickel, phosphorus, lead, antimony, selenium, tin, vanadium, and zinc. Because of the lack of detail in the statistics it is not possible to do a rigorous meta-analysis. What stands out is that almost every study had subjects with seemingly large amounts of at least one of the metals. The significance of metals in meconium is not clear beyond an indication of exposure although some studies have correlated metals in meconium to a number of adverse outcomes. A number of outstanding questions have been identified that, if resolved, would greatly increase the utility of meconium analysis for assessment of long-term gestational metals exposures. Among these are questions of the developmental and long-term significance of metals detected in meconium, the kinetics and interactions among metals in maternal and fetal compartments and questions on best methods for meconium analyses.

Relative source contributions for perchlorate exposures in a lactating human cohort.

Perchlorate is an iodine-uptake inhibitor and common contaminant of food and drinking water. Understanding the amount of perchlorate exposure occurring through non-water sources is essential for accurate estimates of human exposure levels, and establishment of drinking water limits for this pervasive contaminant. The study objective was to determine the amount of perchlorate intake derived from diet rather than water. Subjects provided drinking water samples, detailed fluid-intake records, 24h urine collections and four milk samples for nine days. Samples were analyzed for perchlorate by isotope dilution ion chromatography-tandem mass spectrometry. Amounts of perchlorate derived from drinking water and dietary sources were calculated for each individual. Water of local origin was found to contribute a minor fraction of perchlorate intake. Estimated fraction intake from drinking water ranged from 0 to 36%. The mean and median dose of perchlorate derived from non-water sources by lactating women was 0.18 µg/kg/day (range: 0.06 to 0.36 µg/kg/day.) Lactating women consumed more fluid (mean 2.424 L/day) than has been assumed in recent risk assessments for perchlorate. The data reported here indicate that lactating women may be exposed to perchlorate through dietary sources at markedly higher levels than estimated previously. Exposures to perchlorate from non-water sources may be higher than recent estimates, including those used to develop drinking water standards.

Breastfed infants metabolize perchlorate.

Bifidobacteria are the dominant intestinal bacteria in breastfed infants. It is known that they can reduce nitrate. Although no direct experiments have been conducted until now, inferred pathways for Bifidobacterium bifidum include perchlorate reduction via perchlorate reductase. We show that when commercially available strains of bifidobacteria are cultured in milk, spiked with perchlorate, perchlorate is consumed. We studied 13 breastfed infant-mother pairs who provided 43 milk samples and 39 infant urine samples, and 5 formula-fed infant-mother pairs who provided 21 formula samples and 21 infant urine samples. Using iodine as a conservative tracer, we determined the average urinary iodine (UI) to milk iodine (MI) concentration ratio to be 2.87 for the breastfed infants. For the same samples, the corresponding perchlorate concentration ratio was 1.37 (difference significant, p < 0.001), indicating that perchlorate is lost. For the formula fed infant group the same ratios were 1.20 and 1.58; the difference was not significant (p = 0.68). However, the small number of subjects in the latter group makes it more difficult to conclude definitively whether perchlorate reduction does or does not occur.

Perchlorate, iodine supplements, iodized salt and breast milk iodine content.

This study was undertaken to determine if increasing maternal iodine intake through single dose tablets will decrease breast milk concentrations of the iodine-uptake inhibitor, perchlorate, through competitive inhibition. We also sought to determine if the timing of supplementation influences the fraction of iodine excreted in milk versus urine and to compare the effectiveness of iodized salt as a means of providing iodine to breastfed infants. Thirteen women who did not use supplements, seven of whom used iodized salt and six of whom used non-iodized salt, submitted four milk samples and a 24-h urine collection daily for three days. Women repeated the sampling protocol for three more days during which ~150µg of iodine were taken in the evening and again for three days with morning supplementation. Samples were analyzed using isotope-dilution inductively-coupled plasma-mass spectrometry for iodine and isotope-dilution ion chromatography-tandem mass spectrometry for perchlorate. No statistically significant differences were observed in milk iodine or perchlorate concentrations during the two treatment periods. Estimated perchlorate intake was above the U.S. National Academy of Sciences suggested reference dose for most infants. Single daily dose iodine supplementation was not effective in decreasing milk perchlorate concentrations. Users of iodized salt had significantly higher iodine levels in milk than non-users. Iodized salt may be a more effective means of iodine supplementation than tablets.

Trace iodine quantitation in biological samples by mass spectrometric methods: the optimum internal standard.

Accurate quantitation of iodine in biological samples is essential for studies of nutrition and medicine, as well as for epidemiological studies for monitoring intake of this essential nutrient. Despite the importance of accurate measurement, a standardized method for iodine analysis of biological samples is yet to be established. We have evaluated the effectiveness of (72)Ge, (115)In, and (129)I as internal standards for measurement of iodine in milk and urine samples by induction coupled plasma mass spectrometry (ICP-MS) and of (35)Cl(18)O(4)(-), (129)I(-), and 2-chlorobenzenesulfonate (2-CBS) as internal standards for ion chromatography-tandem mass spectrometry (IC-MS/MS). We found recovery of iodine to be markedly low when IC-MS/MS was used without an internal standard. Percent recovery was similarly low using (35)Cl(18)O(4) as an internal standard for milk and unpredictable when used for urine. 2-Chlorobenzebenzenesulfonate provided accurate recovery of iodine from milk, but overestimated iodine in urine samples by as much as a factor of 2. Percent recovery of iodine from milk and urine using ICP-MS without an internal standard was approximately 120%. Use of (115)In predicted approximately 60% of known values for both milk and urine samples. (72)Ge provided reasonable and consistent percent recovery for iodine in milk samples (approximately 108%) but resulted in approximately 80% recovery of iodine from urine. Use of (129)I as an internal standard resulted in excellent recovery of iodine from both milk and urine samples using either IC-MS/MS and ICP-MS.

Automated measurement of urinary creatinine by multichannel kinetic spectrophotometry.

Urinary creatinine analysis is required for clinical diagnosis, especially for evaluation of renal function. Creatinine adjustment is also widely used to estimate 24-h excretion from spot samples. Few convenient validated approaches are available for in-house creatinine measurement for small- to medium-scale studies. Here we apply the Jáffe reaction to creatinine determination with zone fluidic multichannel kinetic spectrophotometry. Diluted urine sample and reagent, alkaline picric acid, were mixed by a computer-programmed dispenser and rapidly delivered to a four-channel detection cell. The absorbance change was monitored by a flow-through light-emitting diode-photodiode-based detector. Validation results against high-performance liquid chromatography-ultraviolet (HPLC-UV)/mass spectrometry (MS) are presented. Responses for 10-fold diluted samples were linear within clinically relevant ranges (0-250 mg/L after dilution). The system can analyze 70 samples per hour with a limit of detection of 0.76 mg/L. The relative standard deviation was 1.29% at 100 mg/L creatinine (n=225). Correlation with the HPLC (UV quantitation/MS confirmation) system was excellent (linear, r2=0.9906). The developed system allows rapid, simple, cost-effective, and robust creatinine analysis and is suitable for the analysis of large numbers of urine samples.

Intake of iodine and perchlorate and excretion in human milk.

Perchlorate, thiocyanate, and iodine excretion in urine and milk of 13 breastfeeding women was investigated and the results were interpreted by a model of parallel/competitive transport of these species bythe sodium iodide symporter. For each species i, we assumed physiological homeostasis, where i(T,in) equals the corresponding total excretion in urine and milk (i(e,u) + i(e,m)). The fraction of the total excretion that appeared in milk f(I,m) was measured and ranged from 0.394-0.781, 0.018-0.144, and 0.086-0.464 for perchlorate,thiocyanate, and iodine, respectively. The corresponding median values were 0.541, 0.053, and 0.177, respectively. The selectivity factors of perchlorate over iodide transport, and thiocyanate over iodide transport, defined as f(PC,m)/ f(I,m), and f(SCN,m)/ f(I,m), respectively, were 3.14 +/- 1.20 and 0.27 +/- 0.26 while PC(T,in), SCN(T,in), and I(T,in) among individuals varied 4.9, 5.0, and 8.4x, respectively. These transport selectivities are an order of magnitude lower than those indicated by in vitro studies, suggesting that the impact of both these anions on inhibiting iodide transport in milk may have been overestimated in the extant literature. On the other hand, our results showed that 12 of 13 infants did not have an adequate intake of iodine as defined by the Institute of Medicine and 9 out of 13 infants were likely ingesting perchlorate at a level exceeding the reference dose suggested bythe National Academy of Science panel.

Creatinine adjustment of spot urine samples and 24 h excretion of iodine, selenium, perchlorate, and thiocyanate.

Creatinine (CR) adjustment is widely used for the estimation of urinary 24 h excretion from spot urine samples. We have compared CR-adjusted values for urinary iodine, selenium, perchlorate, and thiocyanate to measured 24 h excretion. The urine samples were collected from a cohort of 14 breastfeeding mothers with both spot samples and 24 h collection, 52 24 h and spot sample pairs where the 24 h CR value fell within the "normal" adult female CR excretion range of 0.6-1.6 g/day were considered for this analysis. In addition, a nonlactating female and a male subject provided all micturitions for 1 and 5 days, respectively. Creatinine was analyzed with a Jáffe reaction-based automated analyzer. Iodine and selenium were determined with induction coupled plasma-mass spectrometry (ICP-MS). Perchlorate and thiocyanate were measured with ion chromatography (IC)-isotope dilution tandem mass spectrometry (MS/MS). Creatinine-adjusted values were poor substitutes of the actual 24 h excretion values (average deviation +/-69, 78, 105, and 104% for iodine, selenium, perchlorate, and thiocyanate, respectively.). Over a 5 day period, the 24 h iodine excretion predicted based on creatinine adjustment of spot samples for the same individual deviated between -83.5 to +101% from the actual measured value, the minimum absolute error being 2.5%. Creatinine adjustment for estimation of 24 h excretion from spot samples was not effective for iodine, selenium, perchlorate, or thiocyanate.

Fatty acid profile in milk from goats, Capra aegagrus hircus, exposed to perchlorate and its relationship with perchlorate residues in human milk.

Polyunsaturated fatty acids (PUFA) in milk are vital for normal growth and development of infant mammals. Changes in fatty acid composition were observed in milk fat from goats dosed with perchlorate (0.1 and 1 mg/kg body weight/day) for 31 days, but the effect was not persistent. Adaptation may be induced in these goats to compensate for the perchlorate effect. In an analysis of fatty acid composition in human milk samples, a weak negative correlation was observed between perchlorate concentrations and total PUFA in 38 human milk samples.

Temporal patterns in perchlorate, thiocyanate, and iodide excretion in human milk.

BACKGROUND: Perchlorate and thiocyanate interfere with iodide uptake at the sodium-iodide symporter and are potential disruptors of thyroid hormone synthesis. Perchlorate is a common contaminant of water, food, and human milk. Although it is known that iodide undergoes significant diurnal variations in serum and urinary excretion, less is known about diurnal variations of milk iodide levels. OBJECTIVES: Variability in perchlorate and thiocyanate excretion in human milk has not been examined. Our objective was to determine variability of perchlorate, thiocyanate, and iodide in serially collected samples of human milk. METHODS: Ten lactating women were asked to collect six milk samples on each of 3 days. As an alternative, subjects were asked to collect as many milk samples as comfortably possible over 3 days. Samples were analyzed for perchlorate, iodide, and thiocyanate by ion chromatography coupled with mass spectrometry. RESULTS: Individual perchlorate, iodide, and thiocyanate levels varied significantly over time; there was also considerable variation among individuals. The iodide range, mean +/- SD, and median for all samples (n = 108) were 3.1-334 microg/L, 87.9 +/- 80.9 microg/L, and 55.2 microg/L, respectively. The range, mean +/- SD, and median of perchlorate in all samples (n = 147) were 0.5-39.5 microg/L, 5.8 +/- 6.2 microg/L, and 4.0 microg/L. The range, mean +/- SD, and median of thiocyanate in all samples (n = 117) were 0.4 -228.3 microg/L, 35.6 +/- 57.9 microg/L, and 5.6 microg/L. The data are not symmetrically distributed; the mean is higher than the median in all cases. CONCLUSIONS: Iodine intake may be inadequate in a significant fraction of this study population. Perchlorate and thiocyanate appear to be common in human milk. The role of these chemicals in reducing breast milk iodide is in need of further investigation.

Perchlorate in the United States. Analysis of relative source contributions to the food chain.

Perchlorate has been considered by some a potential threat to human health, especially to developing infants and children because it may inhibit iodide uptake by the sodium iodide symporter (NIS) of the thyroid. In the United States, during the last several decades, environmental perchlorate has had three recognized sources stemming from (a) its use as an oxidizer (including in rocket propellants), (b) its presence in Chilean nitrate fertilizer (CNF), and (c) natural production. An analysis of the relative source strengths and how they may influence entry into the food chain has not been conducted. Averaged over the last --60 years, we estimate that the source strengths have been (a) 10.6, (b) 0.75, and (c) 0.13-0.64 Gg/y for the United States as a whole. Of this, while (b) and (c) represent actual dispersed amounts, the figure in (a) is the amount of perchlorate produced and only a fraction (f) of it has been dispersed and often in a more localized fashion. In addition, dispersal of (b) has taken place only over agricultural land. Considering that the total land area in the United States is 5.5 x the arable land area, in terms of incorporation into the food chain,the figure cited in (b) has a proportionately greater impact. Most estimates of fwill thus suggest that over the considered period, the contribution of CNF to incorporation of perchlorate in the food chain has likely been comparable to oxidizer perchlorate, with natural production being a lesser source. Fireworks presently constitute a potentially important source of increasing importance but a quantitative impact cannotyet be assessed.

Environmental perchlorate: why it matters.

The only known mechanism of toxicity for perchlorate is interference with iodide uptake at the sodium-iodide symporter (NIS). The NIS translocates iodide across basolateral membranes to the thyroid gland so it can be used to form thyroid hormones (TH). NIS is also expressed in the mammary gland during lactation, so that iodide can be transferred from a mother to her child. Without adequate iodide, an infant cannot produce sufficient TH to meet its developmental needs. Effects expected from perchlorate are those that would be seen in conditions of hypothyroidism or hypothyroxinemia. The probability of a permanent adverse effect is greatest during early life, as successful neurodevelopment is TH-dependent. Study of perchlorate risk is complicated by a number of factors including thyroid status of the mother during gestation, thyroid status of the fetus, maternal and infant iodine intake, and exposure of each to other TH-disrupting chemicals. Perhaps the greatest standing issue, and the issue most relevant to the field of analytical chemistry, is the simple fact that human exposure has not been quantified. This review will summarize perchlorate's potential to adversely affect neurodevelopment. Whether current environmental exposures to perchlorate contribute to neuro-impairment is unknown. Risks posed by perchlorate must be considered in conjunction with iodine intake.

Sample processing method for the determination of perchlorate in milk.

In recent years, many different water sources and foods have been reported to contain perchlorate. Studies indicate that significant levels of perchlorate are present in both human and dairy milk. The determination of perchlorate in milk is particularly important due to its potential health impact on infants and children. As for many other biological samples, sample preparation is more time consuming than the analysis itself. The concurrent presence of large amounts of fats, proteins, carbohydrates, etc., demands some initial cleanup; otherwise the separation column lifetime and the limit of detection are both greatly compromised. Reported milk processing methods require the addition of chemicals such as ethanol, acetic acid or acetonitrile. Reagent addition is undesirable in trace analysis. We report here an essentially reagent-free sample preparation method for the determination of perchlorate in milk. Milk samples are spiked with isotopically labeled perchlorate and centrifuged to remove lipids. The resulting liquid is placed in a disposable centrifugal ultrafilter device with a molecular weight cutoff of 10 kDa, and centrifuged. Approximately 5-10 ml of clear liquid, ready for analysis, is obtained from a 20 ml milk sample. Both bovine and human milk samples have been successfully processed and analyzed by ion chromatography-mass spectrometry (IC-MS). Standard addition experiments show good recoveries. The repeatability of the analytical result for the same sample in multiple sample cleanup runs ranged from 3 to 6% R.S.D. This processing technique has also been successfully applied for the determination of iodide and thiocyanate in milk.

Perchlorate and iodide in dairy and breast milk.

Perchlorate inhibits iodide uptake and may impair thyroid and neurodevelopment in infants. Recently, we unambiguously identified the presence of perchlorate in all seven brands of dairy milk randomly purchased from grocery stores in Lubbock, TX. How widespread is perchlorate in milk? Perchlorate in 47 dairy milk samples from 11 states and in 36 human milk samples from 18 states were measured. Iodide was also measured in a number of the samples. Perchlorate was detectable in 81 of 82 samples. The dairy and breast milk means were, respectively, 2.0 and 10.5 microg/L with the corresponding maximum values of 11 and 92 microg/L. Perchlorate is present in virtually all milk samples, the average concentration in breast milk is five times higher than in dairy milk. Although the number of available measurements are few at this point, for breast milk samples with a perchlorate content greater than 10 microg/L, the iodide content is linearly correlated with the inverse of the perchlorate concentration with a r2 of >0.9 (n = 6). The presence of perchlorate in the milk lowers the iodide content and may impair thyroid development in infants. On the basis of limited available data, iodide levels in breast milk may be significantly lower than it was two decades ago. Recommended iodine intake by pregnant and lactating women may need to be revised upward.

Perchlorate in milk.

Perchlorate was unambiguously detected by ion chromatography-suppressed conductivity (IC-CD) and/or ion chromatography-electrospray mass spectrometry (IC-MS) in seven of seven supermarket milk samples bought randomly in Lubbock, TX. Quantitation by IC-MS and IC-suppressed conductivity detection in conjunction with a preconcentration-preelution method provided comparable results. With a sample cleanup procedure that involved protein removal by ethanol and sequential passage though activated alumina and C-18 silica, the limit of detection for perchlorate in milk was 0.5 microg/L. The levels found ranged from 1.7 to 6.4 microg/L. An evaporated milk sample contained perchlorate at 1.1 +/- 0.6 microg/L level, while we did not find detectable levels in a reconstituted powdered milk sample.