Trace element analysis of human seminal plasma: A cautionary tale of preanalytical variation and use of non-traditional matrices in human biomonitoring studies.

Ensuring harmonization of (ultra-)trace element measurements in non-traditional matrices is a particular analytical challenge that is highlighted in this work for seminal plasma as part of the developmental core at the Wadsworth Center Human Health Exposure Analysis Resource Targeted Laboratory. Seminal plasma was collected from 39 male partners of women undergoing in vitro fertilization and analyzed by inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) following deproteinization with concentrated HNO3. Validation was accomplished using: 1) two aqueous NIST SRMs; 2) a seminal plasma QC pool, characterized via standard additions; 3) standard additions on a subset of samples; and 4) sample duplicates. Agreement with NIST certified or reference values were obtained to within ±15% for the SRMs, and agreement between aqueous calibration values and standard additions values agreed to within ±10-20% for all elements. Standard additions of seminal plasma samples revealed varying matrix effects for Cu and Cr that were not found for the pooled samples. Duplicate analyses agreed to within ±10-30% depending on element. A potential source of contamination in colloidal silica used for processing seminal plasma was identified that requires further study. Comparisons with literature indicate lack of consensus for As, Cd, Cr, Mn, Pb, and V content in seminal plasma. Further work is needed to improve harmonization of future studies.

Bone Mineral Composition Among Long-Term Parenteral Nutrition Patients: Postmortem Assessment of Calcium, Phosphorus, Magnesium, and Select Trace Elements.

BACKGROUND: Patients receiving long-term parenteral nutrition (PN) treatment are at risk of developing metabolic bone diseases (MBDs). The bone compartment serves as a repository for a range of metal(loid)s that are administered intravenously to patients via PN solutions. Thus, the mineral composition of patient bones may be linked to the development of MBDs in this group. METHODS: We measured 12 elements in bone samples obtained post mortem from 7 long-term (2-21 years) PN patients and 18 control bones obtained from hip/knee replacement surgery. The samples were cleaned, digested, and subsequently analyzed using a method based on inductively coupled plasma tandem mass spectrometry. RESULTS: Compared with the control group, bones obtained from PN patients were significantly (P < 0.05) depleted in calcium (Ca), phosphorus (P), magnesium (Mg), chromium, and strontium and enriched in manganese (Mn), zinc, barium, cadmium (Cd), and uranium (U). No differences were observed for cobalt or lead. CONCLUSIONS: Depletion of major components of bone mineral (Ca, P, and Mg) and enrichment in known toxicants (Cd, Mn, U) are concerns for PN patients.

Ultra-trace element analysis of human follicular fluid by ICP-MS/MS: pre-analytical challenges, contamination control, and matrix effects.

Follicular fluid (FF), which is the fluid that envelops the developing oocyte (egg cell) in the ovary, can be analyzed to assess trace element content as well as to determine potential exposure to toxic elements in women seeking in vitro fertilization (IVF) treatment. Such measurements may be useful in establishing associations with potential adverse effects on oocyte viability and subsequent pregnancy outcomes. The principal goal of this study was to leverage the next generation of inorganic mass spectrometry based on ICP-MS/MS to address the numerous analytical challenges of (ultra-)trace element analysis of human FF specimens. Ultra-trace element measurements are defined by the Clinical Laboratory Standards Institute as fluid concentrations below 10 µg L-1 or tissue mass fractions below 1 µg g-1. Stringent pre-analytical procedures were developed to minimize exogenous contamination during FF specimen collection and storage in a prospective study of 56 women seeking IVF treatment. ICP-MS/MS instrumental parameters were carefully optimized, and the method validated for 11 biologically important elements that included 4 at trace levels (Cu, Se, Sr, and Zn) and 7 at ultra-trace levels (As, Cd, Co, Mo, Mn, Hg, and Pb). Method limits of detection (LODs) for ultra-trace elements varied from 5.6 ng L-1 for Cd to 0.11 µg L-1 for Mo. A total of 197 human FF specimens were analyzed using the proposed ICP-MS/MS method with 84% of specimens detectable for Pb and 100% detectable for Co, Cu, Mn, Mo, Sr, and Zn. The method based on ICP-MS/MS was compared to a previous method developed for FF using SF-ICP-MS.

An assessment of exposure to rare earth elements among patients receiving long-term parenteral nutrition.

Patients receiving long-term parenteral nutrition (PN) are exposed to potentially toxic elements, which may accumulate in bone. Bone samples collected from seven PN patients (average = 14 years) and eighteen hip/knee samples were analyzed for Al as part of a previous investigation. Yttrium was serendipitously detected in the PN bone samples, leading to the present investigation of rare earth elements (REEs). A method for quantitating fifteen REEs in digested bone was developed based on tandem ICP-MS (ICP-MS/MS) to resolve spectral interferences. The method was validated against nine biological reference materials (RMs) for which assigned values were available for most REEs. Values found in two NIST bone SRMs (1400 Bone Ash and 1486 Bone Meal) compared favorably to those reported elsewhere. Method detection limits ranged from 0.9 ng g-1 (Tm) to 5.8 ng g-1 (Y). Median REE values in the PN patient group were at least fifteen times higher than the "control" group, and exceeded all previously reported data for eleven REEs in human bones. REE content in PN bones normalized to the Earth's upper crust revealed anomalies for Gd in two patients, likely from exposure to Gd-containing contrast agents used in MRI studies. A retrospective review of the medical record for one patient revealed an almost certain case of nephrogenic systemic fibrosis, associated with Gd exposure. Analysis of two current PN formulations showed traces of REEs with relative abundances similar to those found in the PN bones, providing convincing evidence that PN solutions were the primary source of REEs in this population.

Abundance and Significance of Iron, Zinc, Copper, and Calcium in the Hearts of Patients With Friedreich Ataxia.

Cardiomyopathy is a frequent cause of death in patients with Friedreich ataxia (FA), and a characteristic pathological feature is the focal accumulation of iron (Fe) in cardiomyocytes. This restricted localization of the metal contrasts with the diffuse cardiac Fe overload in hemochromatosis and transfusion siderosis. Nevertheless, heart Fe in FA contributes to cardiomyocyte necrosis, inflammation, and scarring as the disease progresses. A putative mechanism of cardiomyopathy in FA is Fe-mediated oxidative damage. Two other transition metals zinc (Zn) and copper (Cu), are diffusely distributed throughout normal hearts and the hearts of patients with FA. The myocardium in FA is also prone to deposits of calcium in the form of scattered concretions. In this study, heart tissues (left and right ventricular walls and ventricular septum) of 23 patients with genetically confirmed FA and 8 normal controls were obtained at autopsy and analyzed for Fe, Zn, Cu, and calcium. The principal assay methods were inductively coupled plasma optical emission spectrometry and plasma mass spectrometry. Total levels of Fe in bulk extracts were not significantly higher than normal, and the concentrations of Zn also remained in the normal range. Cu levels, however, were significantly lower in FA. In conclusion, the decrease of Cu may be important in consideration of the potential benefit of Cu supplements in FA cardiomyopathy.

The pathogenesis of cardiomyopathy in Friedreich ataxia.

Friedreich ataxia (FA) is an autosomal recessive disease with a complex neurological phenotype, but the most common cause of death is heart failure. This study presents a systematic analysis of 15 fixed and 13 frozen archival autopsy tissues of FA hearts and 10 normal controls (8 frozen) by measurement of cardiomyocyte hypertrophy; tissue frataxin assay; X-ray fluorescence (XRF) of iron (Fe) and zinc (Zn) in polyethylene glycol-embedded samples of left and right ventricular walls (LVW, RVW) and ventricular septum (VS); metal quantification in bulk digests by inductively-coupled plasma optical emission spectrometry (ICP-OES); Fe histochemistry; and immunohistochemistry and immunofluorescence of cytosolic and mitochondrial ferritins and of the inflammatory markers CD68 and hepcidin. FA cardiomyocytes were significantly larger than normal and surrounded by fibrotic endomysium. Frataxin in LVW was reduced to less than 15 ng/g wet weight (normal 235.4 ± 75.1 ng/g). All sections displayed characteristic Fe-reactive inclusions in cardiomyocytes, and XRF confirmed significant regional Fe accumulation in LVW and VS. In contrast, ICP-OES analysis of bulk extracts revealed normal total Fe levels in LVW, RVW, and VS. Cardiac Zn remained normal by XRF and assay of bulk digests. Cytosolic and mitochondrial ferritins exhibited extensive co-localization in cardiomyocytes, representing translational and transcriptional responses to Fe, respectively. Fe accumulation progressed from a few small granules to coarse aggregates in phagocytized cardiomyocytes. All cases met the "Dallas criteria" of myocarditis. Inflammatory cells contained CD68 and cytosolic ferritin, and most also expressed the Fe-regulatory hormone hepcidin. Inflammation is an important factor in the pathogenesis of FA cardiomyopathy but may be more evident in advanced stages of the disease. Hepcidin-induced failure of Fe export from macrophages is a likely contributory cause of damage to the heart in FA. Frataxin replacement and anti-inflammatory agents are potential therapies in FA cardiomyopathy.

Toxic metals in seminal plasma and in vitro fertilization (IVF) outcomes.

We measured toxic metals in seminal plasma collected from 30 men using vitro fertilization (IVF), to evaluate associations with semen quality and IVF outcomes. A doubling in Hg-adjusted Pb concentration was associated with 47% lower total motile sperm. Positive associations were suggested for Hg with pregnancy and live birth, adjusted for Cd or Pb. A negative association was suggested for Hg-adjusted Cd with pregnancy. These data add to evidence indicating that toxic metals impact IVF.

Excessive aluminum accumulation in the bones of patients on long-term parenteral nutrition: postmortem analysis by electrothermal atomic absorption spectrometry.

BACKGROUND: Aluminum (Al) contamination of parenteral nutrition (PN) solutions remains a concern for long-term PN patients. Al accumulates particularly in bone. Excessive exposure to Al may result in increased Al body burden and impaired bone formation and mineralization, leading to bone disease. Although the U.S. Food and Drug Administration (FDA) has limited Al contamination in large-volume parenteral solutions, small-volume parenterals may still contribute considerable amounts of Al to PN solutions. The goal of this study is to determine whether or not long-term adult PN patients remain at risk for increased bone Al accumulation. METHODS: We measured Al accumulation in autopsy bones from 7 patients who had received PN for 2-21 years and compared bone Al levels with those in living control patients undergoing hip or knee replacement. Electrothermal atomic absorption spectrometry was used for bone Al measurements. RESULTS: When compared with bone Al content in controls, markedly elevated Al levels (P < .0001) were found in the bones of all but 1 patient, who received PN for only 2 years before death. Even greater Al accumulation was found for PN patients who developed late renal impairment (P = .0159). CONCLUSIONS: We conclude that long-term adult PN patients continue to be at risk for Al toxicity.

Associations between toxic metals in follicular fluid and in vitro fertilization (IVF) outcomes.

PURPOSE: We previously reported associations between trace concentrations of Hg, Cd and Pb in blood and urine and reproductive outcomes for women undergoing in-vitro fertilization (IVF). Here we assess measurements in single follicular fluid (FF) specimens from 46 women as a presumably more relevant marker of dose for reproductive toxicity. METHODS: FF specimens were analyzed for Hg, Cd and Pb using sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS). Variability sources were assessed by nested ANOVA. Multivariable regression was used to evaluate associations for square root transformed metals with IVF outcomes, adjusting for confounders. RESULTS: An inverse association is detected for FF Pb and fertilization (relative risk (RR) = 0.68, P = 0.026), although positive for Cd (RR = 9.05, P = 0.025). While no other statistically significant associations are detected, odds ratios (OR) are increased for embryo cleavage with Hg (OR = 3.83, P = 0.264) and Cd (OR = 3.18, P = 0.644), and for embryo fragmentation with Cd (OR = 4.08, P = 0.586) and Pb (OR = 2.22, P = 0.220). Positive estimates are observed for Cd with biochemical (RR = 19.02, P = 0.286) and clinical pregnancies (RR = 38.80, P = 0.212), yet with very low precision. CONCLUSIONS: We have identified associations between trace amounts of Pb and Cd in FF from a single follicle, and oocyte fertilization. Yet, the likelihood of biological variation in trace element concentrations within and between follicles, coupled with levels that are near the limits of detection suggest that future work should examine multiple follicles using a 'one follicle-one oocyte/embryo' approach. A larger study is merited to assess more definitively the role that these environmental factors could play with respect to egg quality in IVF programs.

A study of the distribution of aluminium in human placental tissues based on alkaline solubilization with determination by electrothermal atomic absorption spectrometry.

Aluminium (Al) is a nonessential element known to induce neurotoxic effects, such as dialysis dementia, in patients on hemodialysis, with compromised kidney function. The role of Al in the progression of some neurodegenerative diseases, such as Alzheimer's disease (AD), is controversial, and remains unclear. The effects of Al on other vulnerable populations, such as fetuses and infants, have been infrequently studied. In the present study, Al has been measured in human placenta samples, comprising ∼160 each of placenta bodies, placenta membranes, and umbilical cords, using electrothermal atomic absorption spectrometry (ETAAS) after atmospheric pressure digestion with tetramethylammonium hydroxide (TMAH) and ethylenediaminetetraacidic acid (EDTA). The sensitivity, or characteristic mass (m(0)), for Al at the 309.3-nm line was found to be 30 ± 4 pg. The instrumental detection limit (IDL) (3s) for Al in solution was calculated as 0.72 µg L(-1) while the method detection limit (MDL) (3s) was 0.25 µg g(-1). Accuracy was assessed through analysis of quality control (QC) materials, including certified reference materials (CRMs), in-house reference materials (RMs), and spike recovery experiments, of varying matrices. Placental tissue analyses revealed geometric mean concentrations of approximately 0.5 µg g(-1) Al in placenta bodies (n = 165) and membranes (n = 155), while Al concentrations in umbilical cords (n = 154) were about 0.3 µg g(-1). Al was detected in 95% of placenta bodies, and 81% of placenta membranes, but only in 46% of umbilical cords.