Associations between follicular fluid trace elements and ovarian response during in vitro fertilization.

INTRODUCTION: Exposure to trace elements has been associated with ovarian response in experimental studies. We conducted a hypothesis-generating study of associations between ovarian follicular fluid (FF) trace elements and measures of ovarian response among women using in vitro fertilization (IVF). METHODS: We collected ovarian FF specimens from 56 women. We determined concentrations (µg/L) of 11 trace elements using inductively coupled plasma-tandem mass spectrometry. We estimated associations between women's FF trace elements per interquartile range difference, and measures of ovarian response using linear (peak estradiol (E2), baseline anti-mullerian hormone (AMH), and follicle stimulating hormone (FSH)) and negative binomial (baseline antral follicle count (AFC) and oocyte count) regression, adjusting for confounding factors. We used principal component analysis (PCA) to estimate the associations of the FF trace elements mixture. We also explored FF oxidative stress enzymes as causal mediators of the associations. RESULTS: Higher FF cobalt was associated with greater peak E2 (mean difference = 351.48 pg/mL; 95%CI: 21.76, 724.71) and AFC (rate ratio = 1.14; 95%CI: 1.01, 1.28), and higher FF copper was associated with greater peak E2 (mean difference = 335.66 pg/mL; 95%CI: 81.77, 753.08) and oocyte count (rate ratio = 1.19; 95%CI: 1.02, 1.43). Higher FF mercury was also associated with greater peak E2 (mean difference = 410.70 pg/mL; 95%CI: 61.90, 883.39). Higher FF lead was associated with lesser AFC (rate ratio = 0.85; 95%CI: 0.73, 0.98). Using PCA, the mixture of Sr, Hg, and As was associated with higher peak estradiol, AFC, and oocyte count. FF glutathione peroxidase, paraoxonase, and arylesterase activities were inconsistent mediators of the associations, but the effect estimates were imprecise. CONCLUSION: Our results suggest that essential and non-essential trace elements in FF were associated with ovarian response during IVF.

Placental cellular composition and umbilical cord tissue metal(loid) concentrations: A descriptive molecular epidemiology study leveraging DNA methylation.

The placenta is a mixture of cell types, which may regulate maternal-fetal transfer of exogenous chemicals or become altered in response to exposures. We leveraged placental DNA methylation to characterize major constituent cell types and applied compositional data analysis to test associations with non-essential metal(loid)s measured in paired umbilical cord tissue (N = 158). Higher proportions of syncytiotrophoblasts were associated with lower arsenic, whereas higher proportions of Hofbauer cells were associated with higher cadmium concentrations in umbilical cords. These findings suggest that placental cellular composition influences amounts of metal(loid)s transferred to the fetus or that prenatal exposures alter the placental cellular makeup.

Metal mixtures modeling identifies birth weight-associated gene networks in the placentas of children born extremely preterm.

Prenatal exposure to toxic metals is linked to numerous adverse birth and later-in-life outcomes. These outcomes are tied to disrupted biological processes in fetal-derived tissues including the placenta and umbilical cord yet the precise pathways are understudied in these target tissues. We set out to examine the relationship between metal concentrations in umbilical cord and altered gene expression networks in placental tissue. These novel relationships were investigated in a subset of the Extremely Low Gestational Age Newborn (ELGAN) cohort (n = 226). Prenatal exposure to 11 metals/metalloids was measured using inductively coupled plasma tandem-mass spectrometry (ICP-MS/MS) in cord tissue, ensuring passage through the placental barrier. RNA-sequencing was used to quantify >37,000 mRNA transcripts. Differentially expressed genes (DEGs) were identified with respect to each metal. Weighted gene co-expression analysis identified gene networks modulated by metals. Two innovative mixtures modeling techniques, namely principal components analysis and quantile-based g-computation, were employed to identify genes/gene networks associated with multi-metal exposure. Individually, lead was associated with the strongest genomic response of 191 DEGs. Joint lead and cadmium exposure was related to 657 DEGs, including DNA Methyl Transferase 1 (DNMT1). These genes were enriched for the Eukaryotic Initiation Factor 2 (EIF2) pathway. Four gene networks, each containing genes within a Nuclear Factor kappa-light-chain-enhancer of Activated B Cells (NF-kB)-mediated network, were significantly increased in average expression level in relation to increases in all metal concentrations. All four of these metal mixture-associated gene networks were negatively correlated with important predictors of neonatal health including birth weight, placenta weight, and fetal growth. Bringing together novel methodologies from epidemiological mixtures analyses and toxicogenomics, applied to a unique cohort of extremely preterm children, the present study highlighted critical genes and pathways in the placenta dysregulated by prenatal metal mixtures. These represent potential mechanisms underlying the developmental origins of metal-induced disease.

Prenatal exposure to multiple metallic and metalloid trace elements and the risk of bacterial sepsis in extremely low gestational age newborns: A prospective cohort study.

Background: Prenatal exposures to metallic and metalloid trace elements have been linked to altered immune function in animal studies, but few epidemiologic studies have investigated immunological effects in humans. We evaluated the risk of bacterial sepsis (an extreme immune response to bacterial infection) in relation to prenatal metal/metalloid exposures, individually and jointly, within a US-based cohort of infants born extremely preterm. Methods: We analyzed data from 269 participants in the US-based ELGAN cohort, which enrolled infants delivered at <28 weeks' gestation (2002-2004). Concentrations of 8 trace elements-including 4 non-essential and 4 essential-were measured using inductively coupled plasma tandem mass spectrometry in umbilical cord tissue, reflecting in utero fetal exposures. The infants were followed from birth to postnatal day 28 with bacterial blood culture results reported weekly to detect sepsis. Discrete-time hazard and quantile g-computation models were fit to estimate associations for individual trace elements and their mixtures with sepsis incidence. Results: Approximately 30% of the extremely preterm infants developed sepsis during the follow-up period (median follow-up: 2 weeks). After adjustment for potential confounders, no trace element was individually associated with sepsis risk. However, there was some evidence of a non-monotonic relationship for cadmium, with hazard ratios (HRs) for the second, third, and fourth (highest) quartiles being 1.13 (95% CI: 0.51-2.54), 1.94 (95% CI: 0.87-4.32), and 1.88 (95% CI: 0.90-3.93), respectively. The HRs for a quartile increase in concentrations of all 8 elements, all 4 non-essential elements, and all 4 essential elements were 0.92 (95% CI: 0.68-1.25), 1.19 (95% CI: 0.92-1.55), and 0.77 (95% CI: 0.57-1.06). Cadmium had the greatest positive contribution whereas arsenic, copper, and selenium had the greatest negative contributions to the mixture associations. Conclusions: We found some evidence that greater prenatal exposure to cadmium was associated with an increased the risk of bacterial sepsis in extremely preterm infants. However, this risk was counteracted by a combination of arsenic, copper, and selenium. Future studies are needed to confirm these findings and to evaluate the potential for nutritional interventions to prevent sepsis in high-risk infants.

Toxic elements in follicular fluid adversely influence the likelihood of pregnancy and live birth in women undergoing IVF.

STUDY QUESTION: Are follicular fluid (FF), arsenic (As), mercury (Hg), cadmium (Cd) and lead (Pb) concentrations associated with IVF outcomes among women undergoing IVF? SUMMARY ANSWER: There was a non-linear association between higher FF Hg concentration and a lower likelihood of biochemical pregnancy and live birth. Higher FF Pb concentration was also associated with a lower probability of live birth. WHAT IS KNOWN ALREADY: Previous research suggests that toxic elements may affect fertility among couples conceiving with and without assistance. However, the results have been inconsistent, possibly related in part to exposure misclassification. Very few studies have used ovarian FF to measure toxic elements, as it requires an invasive collection procedure, yet it may offer a more accurate estimate of a biologically effective dose than blood or urine. STUDY DESIGN SIZE DURATION: This is a prospective study of 56 women undergoing IVF, from October 2015 to June 2017. FF was collected for analysis on the day of oocyte retrieval. PARTICIPANTS/MATERIALS SETTING METHODS: As, Cd, Hg and Pb were determined in 197 FF specimens, using inductively coupled plasma tandem mass spectrometry. FF glutathione peroxidase, glutathione reductase, total glutathione-S-transferase, superoxide dismutase, arylesterase and paraoxonase (PON1p) activities were measured using kinetic enzyme assays. MAIN RESULTS AND THE ROLE OF CHANCE: Non-linear associations were detected, in which the probabilities of biochemical pregnancy (P = 0.05) and live birth (P = 0.05) were lower in association with FF Hg greater than ∼0.51 µg/l Hg, adjusted for age, race, cigarette smoking and recent seafood consumption. Higher FF Pb was also associated with a lower likelihood of live birth (relative risk (RR) = 0.68, 95% CI: 0.46, 1.00; P = 0.05). We also found a suggestive, although imprecise, antagonizing mediating effect of PON1p activity on the association between FF Pb and live birth (-28.3%; 95% CI: -358%, 270%). LIMITATIONS REASONS FOR CAUTION: The results should be interpreted judiciously given the limited sample size and difficulty accounting for correlated data in generalized additive models and mediation analyses. Additionally, women undergoing IVF are highly selected with respect to age and socioeconomic status, and so the generalizability of the results may be limited. WIDER IMPLICATIONS OF THE FINDINGS: Overall, the results suggest that FF Hg was associated with a lower likelihood of biochemical pregnancy and live birth, with a potential threshold effect, and that higher FF Pb was associated with a lower probability of live birth. These results may help to guide clinical recommendations for limiting the exposure of patients to Hg and Pb and ultimately improve IVF success rates. STUDY FUNDING/COMPETING INTERESTS: This work was funded in part by the National Institute of Environmental Health Sciences (NIEHS), grant number 1R56ES023886-01, to the University at Albany (M.S.B.), and in part by the National Institute of Environmental Health Sciences (NIEHS), grant number 1U2CES026542-01, to the Wadsworth Center (P.J.P.). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.

Lead uptake into calcified and keratinized compartments of horns from a convenience sample of lead-dosed goats.

Hair and/or nail analyses are sometimes used in biomonitoring studies due to the convenience of sample collection, storage, and transport, as well as the potential to assess past exposures to toxic metals, such as lead (Pb). However, the validity of Pb measurements in these keratinized matrices as biomarkers of absorbed dose remains unclear. The aim of this study was to examine the uptake of Pb into horns harvested postmortem from 11 goats that received a cumulative oral dose of up to 151 g Pb acetate over a period of 1-11 years as part of a long-term blood Pb proficiency testing program. Uptake of Pb into keratinized horn was compared to the corresponding underlying bony horn core, which, as part of the bone compartment, provided a measure of absorbed Pb dose. Two complementary analytical techniques were used to assess Pb: X-Ray Fluorescence (XRF) and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Detectable amounts of Pb were found in all keratinized horn samples (0.45-6.6 µg/g) and in all but one bony core sample (1.4-68 µg/g). In both bony core and keratinized horn samples, Pb accumulation increased with dose over a low-to-moderate cumulative-dose interval, consistent with previous observations, but plateaued at higher doses. Significant associations were observed between Pb in keratinized horn and bony core samples particularly with XRF measurements, which represent the surface bone compartment. These findings provide evidence that Pb is excreted in keratinized tissues but reflects only a small fraction of the absorbed Pb dose, likely transferred from underlying bone tissue.

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.

Quality assurance and harmonization for targeted biomonitoring measurements of environmental organic chemicals across the Children's Health Exposure Analysis Resource laboratory network.

A consortium of laboratories established under the Children's Health Exposure Analysis Resource (CHEAR) used a multifaceted quality assurance program to promote measurement harmonization for trace organics analyses of human biospecimens that included: (1) participation in external quality assurance (EQA)/proficiency testing (PT) programs; (2) analyses of a urine-based CHEAR common quality control (QC) pool with each analytical batch across all participating laboratories; (3) method validation against NIST Standard Reference Materials® (SRMs); and (4) analyses of blinded duplicates and other project-specific QC samples. The capability of five CHEAR laboratories in organic chemical analysis increased across the 4-year period, and performance in the external PT program improved over time - recent challenges reporting >90% analytes with satisfactory performance. The CHEAR QC pools were analyzed for several classes of organic chemicals including phthalate metabolites and environmental phenols by the participating laboratories with every batch of project samples, which provided a rich source of measurement data for the assessment of intra- and inter-laboratory variance. Within-laboratory and overall variabilities in measurements across laboratories were calculated for target chemicals in urine QC pools; the coefficient of variation (CV) was generally below 25% across batches, studies and laboratories and indicated acceptable analytical imprecision. The suite of organic chemicals analyzed in the CHEAR QC pool was broader than those reported for commercially available reference materials. The accuracy of each of the laboratories' methods was verified through the analysis of several NIST SRMs and was, for example, 97 ± 5.2% for environmental phenols and 95 ± 11% for phthalates. Analysis of blinded duplicate samples showed excellent agreement and reliability of measurements. The intra-class correlation coefficients (ICC) for phthalate metabolites analyzed in various batches across three CHEAR laboratories showed excellent reliability (typically >0.90). Overall, the multifaceted quality assurance protocols followed among the CHEAR laboratories ensured reliable and reproducible data quality for several classes of organic chemicals. Increased participation in external PT programs through inclusion of additional target analytes will further enhance the confidence in data quality.

Variability of essential and non-essential trace elements in the follicular fluid of women undergoing in vitro fertilization (IVF).

Both essential and non-essential elements have been associated with female reproductive function in epidemiologic investigations, including among IVF populations. To date, most investigators have used blood or urine to assess biomarkers of exposure, with few employing ovarian follicular fluid (FF). FF may offer a more direct "snapshot" of the oocyte microenvironment than blood or urine, however previous studies report follicle-to-follicle variability in FF constituents that may contribute to exposure misclassification. Our objectives were to investigate sources of trace element variability, to estimate FF biomarker reliability among women undergoing IVF (n = 34), and to determine the minimum number of follicles required to estimate subject-specific mean concentrations. We measured As, Hg, Cd, Pb, Cu, Mn, Se, and Zn in FF samples using inductively coupled plasma tandem mass spectrometry. Inter-subject (between-women) variability contributed most of the variability in FF element concentrations, with ovarian, follicular, and analytical as smaller sources of variability. The proportion of variability attributable to sources between-follicles differed by age, body mass index (BMI), race, and cigarette smoking for Cu, Se, and Zn, by BMI and cigarette smoking for As, by primary infertility diagnosis for Hg, Cu, Se, and Zn, and by ovarian stimulation protocol for Mn and Se. Four to five individual follicles were sufficient to estimate subject-specific mean Cu, Se, and Zn concentrations, while >14 were necessary for As, Hg, Cd, Pb, and Mn. Overall, our results suggest that FF is a suitable source of biomarkers of As and Hg exposure in ovarian follicles. Although limited in size, our study offers the most comprehensive exploration of biological variation in FF trace elements to date and may provide guidance for future studies of ovarian trace element exposures.

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.

Measurement harmonization and traceability for trace element analyses across the Children's Health Exposure Analysis Resource laboratory network.

Harmonization and traceability are related metrological principles that are indispensable to assuring measurement comparability across different biomonitoring studies. The Children's Health Exposure Analysis Resource (CHEAR) was established in 2015 with six laboratories providing environmental exposure measurements on biospecimens. To ensure harmonization across studies for trace elements, CHEAR used a multi-faceted approach that included: 1) an initial interlaboratory validation exercise based on the analysis of certified blood and urine reference materials; 2) frequent participation in an established interlaboratory proficiency program for trace elements; and 3) analysis of a common pool of well-characterized biological reference materials with each analytical batch. Method accuracy and precision were established for each laboratory via analysis of NIST SRM 955c Toxic Elements in Caprine Blood, SRM 2668 Toxic Elements in Frozen Human Urine and SRM 3668 Mercury, Perchlorate, and Iodide in Frozen Human Urine. The differences among the six laboratories for As, Cd, Hg, and Mn in urine and Cd, Hg, and Pb in whole blood were judged to be fit-for-purpose. Interlaboratory performance over a 5-year period demonstrated an improvement in performance, such that for 2018-2019, >99% of challenges for urine As, Cd, Hg, and Mn, and 95% for whole blood Cd, Hg, Pb, and Mn, were found to be satisfactory. The CHEAR common reference materials were analyzed by at least 5 laboratories for 22 elements in urine and 13-14 elements in whole blood, thus providing a rich source of data to assess intra- and inter-run performance. The suite of trace elements with assigned values in both blood and urine matrices are more comprehensive than similar reference materials from other sources, and is reflective of the concentrations necessary to support biomonitoring studies. While some areas for future improvement were identified, significant progress was made to improve harmonization of trace element measurements in biological matrices among the CHEAR network labs.

Seafood consumption is associated with higher follicular fluid arsenic (As) and mercury (Hg) concentrations in women undergoing in vitro fertilization (IVF).

Human exposure to non-essential toxic metals such as cadmium (Cd), mercury (Hg), and lead (Pb), and metalloids such as arsenic (As) commonly occurs through diet. Toxic trace element exposures have been reported in association with fertility and fecundity in epidemiologic studies even at low to moderate levels. While most previous studies employed blood and urine biomarkers of exposure, few have assessed toxic trace elements in ovarian follicular fluid (FF), which surrounds the developing oocyte and hence may better reflect concentrations potentially affecting reproductive outcomes. Our objective was to identify dietary predictors of FF toxic trace elements in n = 56 women (mean age: 38.3 years) undergoing in vitro fertilization (IVF) at the University of California at San Francisco. We determined As, Hg, Cd, and Pb in 197 FF specimens, collected on the day of oocyte retrieval, using inductively coupled plasma tandem mass spectrometry. A comprehensive food frequency questionnaire was used to assess the weekly and annual dietary "patterns" of participants. Consumption of specific seafood items and turkey were correlated with individual FF toxic trace elements. We also found that each unit higher seafood consumption in the past week dominated by mollusks, shrimp, and bass was associated with 60% higher FF As (95% confidence interval (CI): 25%, 105%) and FF Hg (95%CI: 7%, 136%) concentrations. Higher annual seafood consumption dominated by urchin, crab, and trout was associated with 16% higher FF As (95%CI: -2%, 38%) and 31% higher FF Hg (95%CI: 7%, 60%) concentrations per unit intake. No associations were noted between diet and Cd and Pb levels in FF. Overall, our results suggest that higher seafood consumption contributes to elevated levels of As and Hg in FF. These findings are consistent with previous IVF studies that assessed toxic trace element exposures in blood and urine. To our knowledge, this is the first study to report that diet might be a source of As, Hg, Cd, and Pb in FF.

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.

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.