Development of a pregnancy-specific reference material for thyroid biomarkers, vitamin D, and nutritional trace elements in serum.

OBJECTIVES: Matrix differences among serum samples from non-pregnant and pregnant patients could bias measurements. Standard Reference Material 1949, Frozen Human Prenatal Serum, was developed to provide a quality assurance material for the measurement of hormones and nutritional elements throughout pregnancy. METHODS: Serum from non-pregnant women and women in each trimester were bottled into four levels based on pregnancy status and trimester. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods were developed and applied to the measurement of thyroid hormones, vitamin D metabolites, and vitamin D-binding protein (VDBP). Copper, selenium, and zinc measurements were conducted by inductively coupled plasma dynamic reaction cell MS. Thyroid stimulating hormone (TSH), thyroglobulin (Tg), and thyroglobulin antibody concentrations were analyzed using immunoassays and LC-MS/MS (Tg only). RESULTS: Certified values for thyroxine and triiodothyronine, reference values for vitamin D metabolites, VDBP, selenium, copper, and zinc, and information values for reverse triiodothyronine, TSH, Tg, and Tg antibodies were assigned. Significant differences in serum concentrations were evident for all analytes across the four levels (p≤0.003). TSH measurements were significantly different (p<0.0001) among research-only immunoassays. Tg concentrations were elevated in research-only immunoassays vs. Federal Drug Administration-approved automated immunoassay and LC-MS/MS. Presence of Tg antibodies increased differences between automated immunoassay and LC-MS/MS. CONCLUSIONS: The analyte concentrations' changes consistent with the literature and the demonstration of matrix interferences in immunoassay Tg measurements indicate the functionality of this material by providing a relevant matrix-matched reference material for the different stages of pregnancy.

Development of a kelp powder (Thallus laminariae) Standard Reference Material.

A Standard Reference Material (SRM) of seaweed, SRM 3232 Kelp Powder (Thallus laminariae) has been developed to support food and dietary supplement measurements in compliance with the Food Safety Modernization Act (FSMA) and the Dietary Supplement Health and Education Act of 1994 (DSHEA). The material was characterized for nutritional minerals, arsenic species, isomers of vitamin K1, proximates, and toxic elements. Kelp is a rich source of vitamins and minerals, and it is an excellent source of dietary iodine. Kelp also contains a large amount of arsenic, which is toxic as inorganic species but much less so as organic species. To capture the dietary profile of kelp, certified values were issued for As, Ca, Cd, Cr, Cu, Fe, Hg, I, K, Mg, Mn, Mo, Na, Pb, and Zn. Reference values for proximates were assigned. For the first time, a certified value for iodine, reference values for isomers of vitamin K1, and reference values for arsenic species including arsenosugars were assigned in a seaweed. SRM 3232 fills a gap in Certified Reference Materials (CRMs) needed for quality assurance and method validation in the compositional measurements of kelp and similar seaweeds used as food and as dietary supplements. Graphical Absract Arsenic species and isomers of vitamin K1 were determined in the development of SRM 3232 Kelp Powder (Thallus laminariae).

Urinary iodine and stable isotope analysis to examine habitat influences on thyroid hormones among coastal dwelling American alligators.

The American alligator, generally a freshwater species, is known to forage in marine environments despite the lack of a salt secreting gland found in other crocodylids. Estuarine and marine foraging could lead to increased dietary uptake of iodine, a nutrient necessary for the production of thyroid hormones. To explore the influence of dietary iodine on thyroid hormone health of coastal dwelling alligators, we described the seasonal plasma thyroxine and triiodothyronine concentrations measured by radioimmunoassay and urinary iodine (UI) concentrations measured by inductively coupled plasma mass spectrometry. We also analyzed long-term dietary patterns through stable isotope analysis of scute tissue. Snout-to-vent length (SVL) was a significant factor among UI and stable isotope analyses. Large adult males greater than 135cm SVL had the highest UI concentrations but did not display seasonality of thyroid hormones. Alligators under 135 SVL exhibited seasonality in thyroid hormones and a positive relationship between UI and triiodothyronine concentrations. Isotopic signatures provided supporting evidence that large males predominantly feed on marine/estuarine prey whereas females showed reliance on freshwater/terrestrial prey supplemented by marine/estuarine prey. UI measurement provided immediate information that correlated to thyroid hormone concentrations whereas stable isotope analysis described long-term dietary patterns. Both techniques demonstrate that adult alligators in coastal environments are utilizing estuarine/marine habitats, which could alter thyroid hormone physiology.

Examining toxic trace element exposure in American alligators.

Toxic trace element exposure occurs through release of the ubiquitous and naturally occurring elements arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg). The unique environmental conditions of the wetland ecosystems along the southeastern Atlantic coast of the United States lead to the accumulation of Hg which is greater than in most other ecosystems in the country. There are also point sources of As, Cd, and Pb in this region. To effectively monitor trace element concentrations, and consequently the potential human exposure, accessible local sentinel species are needed. In this study, concentrations of As, Cd, Pb, Hg and six other trace elements (Al, Ni, Cu, Zn, Se, Mo) were examined in American alligators (Alligator mississippiensis) from seven wetland sites in South Carolina and Florida and assessed for their utility as a sentinel species for human trace element exposure. Alligators were chosen as a potential sentinel as they share a common exposure with the local human population through their aquatic diet, and they are directly consumed commercially and through recreation hunting in this region. Sex was significantly related to the concentration of Zn, Mo, and Al, but not As, Pb, Hg, Cd, Se, or Cu. Site specific differences in element concentrations were observed for As, Pb, Hg, Cd, Se, Zn, and Mo. Size/age was significantly related to the element Hg and Pb concentrations observed. The observed concentration ranges for the four toxic elements, As (6-156 ng/g), Cd (0.3-1.3 ng/g), Pb (3-4872 ng/g), and Hg (39-2765 ng/g), were comparable to those previously reported in diverse human populations. In this region alligators are hunted recreationally and consumed by the local community, making them a vehicle of direct human toxic element exposure. We propose that the similarity in As, Cd, Pb, and Hg concentrations between alligators observed in this study and humans underscores how alligators can serve as a useful sentinel species for toxic element exposure.

Trace element biodistribution in the American alligator (Alligator mississippiensis).

Routine monitoring of contaminant levels in wildlife is important for understanding chemical exposure and ultimately the link to ecosystem and human health. This is particularly important when the monitored species is recreationally hunted for human consumption. In the southeastern United States, recreational alligator harvesting takes place annually and in locations that are known to be contaminated with environmental pollutants. In this study, we investigated the biodistribution of trace elements in the American alligator (Alligator mississippiensis) from five sites in Florida, USA. These sites are locations where annual recreational alligator harvesting is permitted and two of the sites are identified as having high mercury contamination with human consumption advisories in effect. We utilized routinely collected monitoring samples (blood and scute), a commonly consumed tissue (muscle), and a classically analyzed tissue for environmental contaminants (liver) to demonstrate how the trace elements were distributed within the American alligator. We describe elemental tissue compartmentalization in an apex predator and investigate if noninvasive samples (blood and scute) can be used to estimate muscle tissue concentrations for a subset of elements measured. We found significant correlations for Hg, Rb, Se, Zn and Pb between noninvasive samples and consumed tissue and also found that Hg was the only trace metal of concern for this population of alligators. This study fills a gap in trace elemental analysis for reptilian apex predators in contaminated environments. Additionally, comprehensive elemental analysis of routinely collected samples can inform biomonitoring efforts and consumption advisories.

Global DNA methylation loss associated with mercury contamination and aging in the American alligator (Alligator mississippiensis).

Mercury is a widespread environmental contaminant with exposures eliciting a well-documented catalog of adverse effects. Yet, knowledge regarding the underlying mechanisms by which mercury exposures are translated into biological effects remains incomplete. DNA methylation is an epigenetic modification that is sensitive to environmental cues, and alterations in DNA methylation at the global level are associated with a variety of diseases. Using a liquid chromatography tandem mass spectrometry-based (LC-MS/MS) approach, global DNA methylation levels were measured in red blood cells of 144 wild American alligators (Alligator mississippiensis) from 6 sites with variable levels of mercury contamination across Florida's north-south axis. Variation in mercury concentrations measured in whole blood was highly associated with location, allowing the comparison of global DNA methylation levels across different "treatments" of mercury. Global DNA methylation in alligators across all locations was weakly associated with increased mercury exposure. However, a much more robust relationship was observed in those animals sampled from locations more highly contaminated with mercury. Also, similar to other vertebrates, global DNA methylation appears to decline with age in alligators. The relationship between age-associated loss of global DNA methylation and varying mercury exposures was examined to reveal a potential interaction. These findings demonstrate that global DNA methylation levels are associated with mercury exposure, and give insights into interactions between contaminants, aging, and epigenetics.