The Department of Veterans' Affairs Depleted Uranium Cohort in the Time of COVID-19: Translating a Traditional Surveillance Protocol to a Telehealth Platform.

OBJECTIVE: In 2021, 37 members of a cohort of depleted uranium-exposed Gulf War I veterans were evaluated using a protocol tailored to accommodate COVID-19 safety practices on a telehealth platform. METHODS: Individual elements of the legacy protocol were reviewed for urgency and feasibility of inclusion in a modified, telehealth platform. RESULTS: The redesigned protocol included a participant readiness for telehealth assessment, nurse and physician telehealth visits, collection of usual health questionnaires, and urine collections for exposure monitoring for uranium and other fragment-related metal measures. CONCLUSIONS: Despite some limitations in scope, the telehealth platform permitted a visual "visit" with surveillance participants who expressed a high comfort level with the format. The telehealth platform has apparent utility for occupational surveillance and should be explored as a standard approach for surveillance outside of public health emergencies.

Analysis of umbilical cord tissue as an indicator of in utero exposure to toxic adulterating substances.

In utero drug exposure is a significant public health threat to the well-being and normal development of the neonate. Recently, testing of umbilical cord tissue (UCT) has been employed to measure illicit drug exposure, as drugs used by the mother during the third trimester may be retained in the UCT. Focus has also been given to potential adverse health effects among drug users, resulting from exposure to pharmacologically active adulterants and cutting agents in the street drug supply. The in utero effects of these substances have not been well studied in humans, nor has their presence been demonstrated as a means for assessing adverse health effects in the neonate. Here, we describe the application of a novel test method to analyze UCT for the presence of more than 20 common adulterating/cutting substances via LC/Q-TOF. In total, 300 de-identified UCT samples were analyzed-all had previously tested positive for cocaine or opiates. Generally, the positivity rates of individual compounds were similar between the Cocaine and Opiates Subgroups, apart from levamisole, xylazine, dipyrone (metabolites), and promethazine. Many of the adulterants used in the street drug supply do have legitimate medicinal/therapeutic uses, including several of the compounds most frequently detected in this study. Caffeine and lidocaine were the most frequently identified compounds both individually (>70% each) and in combination with each other. Alternatively, levamisole, an adulterant with no legitimate therapeutic use, was present in 12% of cases. Importantly, this data demonstrates that the detection of traditional drugs of abuse may serve as indicators of potential in utero exposure to toxic adulterating substances during gestation. While there is cause for concern with respect to any unintentional drug exposure, illicit drug use during pregnancy, including uncontrolled dosing, poly-adulterant consumption, and the interactions of these drug mixtures, produces a significant public health threat to the neonate which warrants further study.

Qualitative LC-Q-TOF Analysis of Umbilical Cord Tissue via Data-Dependent Acquisition as an Indicator of In Utero Exposure to Toxic Adulterating Substances.

Toxic adulterants are drug or chemical agents used to add bulk volume to traditional drugs of abuse such as cocaine and heroin. These cutting agents include levamisole, metamizole, noxiptillin, phenacetin and xylazine as well as common legal drugs such as acetaminophen, caffeine, diphenhydramine, lidocaine, quinine, quetiapine and tramadol. Because they possess pharmacological activity they result in exposure of the user, but also in the case of pregnant women, the developing fetus, to potential drug toxicity. We describe the development, validation and implementation of a rapid (48 second sample-to-sample) test based on a qualitative data-dependent liquid chromatography-quadrupole time of flight mass spectrometry method for the analysis of toxic adulterating substances in umbilical cord tissue (UCT) samples. The method provides a means of studying potential in utero exposure to these agents. Library spectra comparison at three different collision energies was used in conjunction with retention time and accurate mass to identify these substances in UCT. Analytically based reporting limits were established to determine positivity rates of adulterants in UCT utilizing a standard addition approach. The method was applied to authentic cocaine and opioid positive UCT to screen for toxic adulterants. There were a total of 82 potential adulterant positives found in a 30-sample cohort of authentic UCT samples, with an average of 2.7 substances per case. Lidocaine was the predominant finding followed by caffeine, and diphenhydramine all of which could result from non-illicit drug exposure, however, there were positives for levamisole, phenacetin, noxiptillin and xylazine none of which are approved in the United States for human therapeutic use. This initial set of data established a preliminary positivity rate of potentially toxic adulterants in UCT samples positive for cocaine or opioid use.

Surveillance of Depleted Uranium-exposed Gulf War Veterans: More Evidence for Bone Effects.

ABSTRACT: Gulf War I veterans who were victims of depleted uranium (DU) "friendly-fire" incidents have undergone longitudinal health surveillance since 1994. During the spring of 2019, 36 members of the cohort were evaluated with a monitoring protocol including exposure assessment for total and isotopic uranium concentrations in urine and a comprehensive review of health outcomes, including measures of bone metabolism and bone mineral density (BMD) determination. Elevated urine U concentrations were observed in cohort members with retained depleted uranium (DU) shrapnel fragments. In addition, a measure of bone resorption, N-telopeptide, showed a statistically significant increase in those in the high DU subgroup, a finding consistent with a statistically significant decrease in bone mass also observed in this high DU subgroup compared to the low DU subgroup. After more than 25 y since first exposure to DU, an aging cohort of military veterans continues to show few U-related health effects in known target organs of U toxicity. The new finding of impaired BMD in the high DU subgroup has now been detected in two consecutive surveillance visits. While this is a biologically plausible uranium effect, it is not reflected in other measures of bone metabolism in the full cohort, which have largely been within normal limits. However, ongoing accrual of the U burden from fragment absorption over time and the effect of aging further impairing BMD suggest the need for future surveillance assessments of this cohort.

The Utility of Spot vs 24-Hour Urine Samples for Metal Determination in Veterans With Retained Fragments.

OBJECTIVES: The objective of this investigation is to explore the utility of using a spot urine sample in lieu of a 24-hour collection in assessing fragment-related metal exposure in war-injured veterans. METHODS: Twenty-four veterans collected each urine void over a 24-hour period in separate containers. Concentrations of 13 metals were measured in each void and in a pooled 24-hour sample using inductively coupled plasma mass spectrometry. To assess the reliability of spot sample measures over time, intraclass correlations (ICCs) were calculated across all spot samples. Lin's concordance correlation coefficient was used to assess agreement between a randomly selected spot urine sample and each corresponding 24-hour sample. RESULTS: In total, 149 spot urine samples were collected. Ten of the 13 metals measured had ICCs more than 0.4, suggesting "fair to good" reliability. Concordance coefficients were more than 0.4 for all metals, suggesting "moderate" agreement between spot and 24-hour concentrations, and more than 0.6 for seven of the 13 metals, suggesting "good" agreement. CONCLUSIONS: Our fair to good reliability findings, for most metals investigated, and moderate to good agreement findings for all metals, across the range of concentrations observed here, suggest the utility of spot urine samples to obtain valid estimates of exposure in the longitudinal surveillance of metal-exposed populations.

Distributions of alcohol use biomarkers including ethanol, phosphatidylethanol, ethyl glucuronide and ethyl sulfate in clinical and forensic testing.

Laboratory tests vary widely in their utility and each test has unique advantages and disadvantages. For the detection of ethanol use and abuse, a variety of direct and indirect markers are available. Alcohol biomarkers provide objective measures for numerous areas of testing including clinical trials, alcohol abuse, postmortem assessment, and drugs of abuse screening. Because the utility of alcohol biomarkers vary depending on the context in which the results will be used, knowing the analogous distribution of results is of value. Herein we report distributions of ethanol in blood, phosphatidylethanol in blood, ethyl glucuronide in urine, and ethyl sulfate in urine for results reported in the last twelve months by our laboratory. Positivity rates were higher for directed analyses when compared to broad screening or panel tests with the highest overall positivity for ethyl glucuronide and ethyl sulfate. The distribution of results for ethyl glucuronide and ethyl sulfate were higher in clinical testing scenarios compared to forensic and a significant correlation between ethyl glucuronide and ethyl sulfate was found consistent with previous reports. Phosphatidylethanol was rarely ordered for forensic use while distributions between routine clinical and clinical trial use were similar. Approximately 21% of all phosphatidylethanol results were in the moderate to chronic alcohol use category. These results provide a summary of four commonly used direct markers for alcohol use with positivity rates and overall quantitative distributions. These data supply insights broken out by various disciplines where applicable providing a concise comparison of results for these markers.

Clinical and Genome-Wide Analysis of Serum Platinum Levels after Cisplatin-Based Chemotherapy.

PURPOSE: Serum platinum is measurable for years after completion of cisplatin-based chemotherapy (CBC). We report the largest investigation of serum platinum levels to date of 1,010 testicular cancer survivors (TCS) assessed 1-35 years after CBC and evaluate genetic contributions to these levels. EXPERIMENTAL DESIGN: Eligible TCS given 300 or 400 (±15) mg/m2 cisplatin underwent extensive audiometric testing, clinical examination, completed questionnaires, and had crude serum platinum levels measured. Associations between serum platinum and various risk factors and toxicities were assessed after fitting a biexponential model adjusted for follow-up time and cumulative cisplatin dose. A genome-wide association study (GWAS) was performed using the serum platinum residuals of the dose and time-adjusted model. RESULTS: Serum platinum levels exceeded the reference range for approximately 31 years, with a strong inverse relationship with creatinine clearance at follow-up (age-adjusted P = 2.13 × 10-3). We observed a significant, positive association between residual platinum values and luteinizing hormone (age-adjusted P = 6.58 × 10-3). Patients with high residual platinum levels experienced greater Raynaud phenomenon than those with medium or low levels (age-adjusted ORhigh/low = 1.46; P = 0.04), as well as a higher likelihood of developing tinnitus (age-adjusted ORhigh/low = 1.68, P = 0.07). GWAS identified one single-nucleotide polymorphism (SNP) meeting genome-wide significance, rs1377817 (P = 4.6 × 10-8, a SNP intronic to MYH14). CONCLUSIONS: This study indicates that residual platinum values are correlated with several cisplatin-related toxicities. One genetic variant is associated with these levels.

Inductively coupled plasma mass spectrometry assay for quantification of free infliximab in serum.

TNF antagonists such as infliximab are effective for the treatment of several inflammatory and autoimmune diseases. Recent clinical studies have advocated the importance of measuring trough infliximab levels to guide treatment decisions. We have developed a novel assay for measuring serum free infliximab levels using inductively coupled plasma-mass spectrometry (ICP-MS). The method involves the incubation of patient serum in wells coated with recombinant TNF, followed by detection with lanthanide-labeled monoclonal anti-human IgG1 and ICP-MS analysis. Full method validation was performed and results for clinical samples tested with the new method were compared with those obtained from a capture ELISA and a cell-based assay. Validation of the ICP-MS assay revealed a lower limit of detection of 0.4 µg/mL in serum. The linear range of quantitation was 1-50 µg/mL. The within-run and between-run precision had a coefficient of variation (CV) of <10%, and the accuracy of the assay had a CV of <15%. In serum samples, the ICP-MS method was devoid of analytical interferences by high levels of hemoglobin, bilirubin and triglycerides. Serum sample results from 123 drug-naïve donors revealed a test cutoff at 0.5 µg/mL. Test results from clinical samples obtained by the ICP-MS method showed strong correlation with both the ELISA and cell-based assay. The ICP-MS methodology presented in this study is a robust method for measuring TNF antagonist serum levels, which makes it well suited for therapeutic drug monitoring in the clinical laboratory.

Association of PTHrP levels in CSF with Alzheimer's disease biomarkers.

BACKGROUND: Parathyroid hormone-related protein (PTHrP) is involved in intracellular calcium regulation, neural cell proliferation and synaptic transmission. To date, no studies have been performed to evaluate the potential of PTHrP concentrations in cerebrospinal fluid (CSF) as a biomarker of brain pathophysiology. In this study we evaluated the association between CSF concentrations of PTHrP with the core CSF biomarkers of Alzheimer's disease (AD). METHODS: PTHrP and calcium were analysed using validated mass spectrometry based methods in a set of CSF samples that tested positive (n = 45) and negative (n = 45) for the AD biomarkers, including total tau protein (T-tau), phosphorylated tau protein (P-tau) and amyloid-ß 42 (Aß42). The measured CSF concentrations of PTHrP and calcium (Ca) were evaluated for association with AD CSF biomarkers. RESULTS: PTHrP and Ca concentrations in CSF samples ranged between 25 and 137 pmol/L and 0.92-1.53 mmol/L, respectively. Higher concentrations of PTHrP were observed in association with increased concentrations of T-tau and P-tau in the AD and the control group; while a stronger correlation was observed in the control group (ρ = 0.6, p < 0.0001; and ρ = 0.72, p < 0.0001, for T-tau and P-tau, respectively). Negative correlation was observed between concentrations of PTHrP and Aß42 in the AD group (ρ = 0.27, p = 0.015). A statistically significantly lower ratio Aß42/PTHrP was observed in the AD group (p < 0.0001). CONCLUSION: In the current study, we observed an association of PTHrP concentrations with concentrations of clinically used CSF biomarkers of AD. Concentrations of PTHrP were positively correlated with concentrations of T-tau and P-tau, suggesting an association with neuronal secretion and function, which is reduced upon progression to AD pathology. Our data suggest potential utility of the Aß42/PTHrP ratio in assessment of AD progression.

Unrecognized Elevations of Toxic Elements in Urine and Blood Highlight the Potential Need for a Broader Approach to Exposure Assessment.

Heavy metals testing remains an ongoing challenge for diagnosing acute or chronic exposure to heavy metals. In this study, we determined the positivity rates of single element and panel testing for toxic elements, and evaluated the potential utility of an expanded detection protocol for screening of toxic element exposures. The retrospective analysis included data from urine (n = 19,343) and blood (n = 196,019) specimens tested using inductively coupled plasma-mass spectrometry (ICP-MS) for arsenic, cadmium, lead and mercury (blood), and arsenic, cadmium, copper, lead, mercury and zinc (urine). Lead industrial monitoring in blood and cadmium exposure in blood and urine were included to represent directed single element ordering. The percent of positive results, defined as results greater than the upper limit of the reference interval was determined. For blood, the highest positivity was observed for lead occupational exposure monitoring (26.2%) whereas for urine, the highest positivity was observed for zinc testing (28.1%). Remarkably, reanalysis using an expanded panel, of 120 blood and 174 urine specimens originally negative identified 42% (50 of 120) of the blood specimens with at least one elevated result and 48% (83 of 174) of the urine specimens with at least one elevated result. Our results indicate that a broad elemental screening panel may help ensure easier identification of elemental exposure and may eliminate the need for additional follow-up sample collections.

Simultaneous Determination of Uranium and Depleted Uranium Isotopic Ratio Using Inductively Coupled Mass Spectrometry.

We have developed and validated a method for the simultaneous quantitative measurement of total uranium (TU) and uranium 235 U/238 U isotopic ratio (UIR) in urine by inductively coupled plasma mass spectrometry (ICP-MS) using a Thermo Scientific iCAP-Q instrument. The performance characteristics of the assay were determined to be in compliance with clinical laboratory standards. The assay was linear in the concentration range of 1.0 to 500.0 ng/liter TU. The method was precise and accurate with limits of detection of 2.5 ng/liter for TU and 9.8 ng/liter for UIR. The accuracy was >93% and the coefficient of variation (% CV) was <5.0% for both TU and UIR. All results were within established guidelines and agreed-upon criteria, and the results fell within the certified range for the reference controls. The method has thus been shown to be effective as a simple, precise, and sensitive analytical technique for testing urine samples. © 2018 by John Wiley & Sons, Inc.

Surveillance results and bone effects in the Gulf War depleted uranium-exposed cohort.

A small group of Gulf War I veterans wounded in depleted uranium (DU) friendly-fire incidents have been monitored in a clinical surveillance program since 1993. During the spring of 2017, 42 members of the cohort were evaluated with a protocol which includes exposure monitoring for total and isotopic uranium concentrations in urine and a comprehensive assessment of health outcomes including measures of bone metabolism, and for participants >50 years, bone mineral density (BMD) determination. Elevated urine U concentrations were observed in cohort members with retained DU shrapnel fragments. Only the mean serum estradiol concentration, a marker of bone metabolism, was found to be significantly different for lower-vs- higher urine U (uU) cohort sub-groups. For the first time, a significant deficit in BMD was observed in the over age 50, high uU sub-group. After more than 25 years since first exposure to DU, an aging cohort of military veterans continues to exhibit few U-related adverse health effects in known target organs of U toxicity. The new finding of reduced BMD in older cohort members, while biologically plausible, was not suggested by other measures of bone metabolism in the full (all ages) cohort, as these were predominantly within normal limits over time. Only estradiol was recently found to display a difference as a function of uU grouping. As BMD is further impacted by aging and the U-burden from fragment absorption accrues in this cohort, a U effect may be clarified in future surveillance visits.

Determination of Thirteen Trace and Toxic Elements in Urine Using Inductively Coupled Mass Spectrometry.

We developed and validated a method for the assessment of thirteen separate trace and toxic elements using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Included elements were as follows: aluminum, chromium, manganese, iron, cobalt, nickel, copper, zinc, arsenic, molybdenum, cadmium, tungsten, and lead. The measurements of all elements in urine samples were conducted using ICAP-Q ICP-MS in a single method. The performance characteristics of the assay were determined according to clinical laboratory standards. The assay was linear in the concentration range of 1.0 to 1000.0 µg/liter for all elements. The method was precise and accurate with limits of quantitation of 1 µg/liter for chromium, manganese, cobalt, nickel, copper, cadmium, tungsten, and lead; 2 µg/liter for iron and arsenic; 5 µg/liter for aluminum; and 50 µg/liter for zinc. This method has successfully been used for the assessment of all thirteen elements included in urine and has been shown to be effective as a simple, precise, and sensitive analytical technique for biological monitoring of urine samples. © 2018 by John Wiley & Sons, Inc.

Lowering the Bar for Mass Spectrometry: A Comparison between Immunoassay and Rapid Time-of-Flight for Presumptive Screening of Drugs in Urine.

BACKGROUND: Immunoassay-based techniques and creatinine quantification have historically been the methods of choice for urine drug screening. Positive presumptive drug screen results are reflexed to more specific, confirmatory testing using gas or liquid chromatography coupled to mass spectrometry. False positives and false negatives with immunoassay techniques are common problems that have substantial down-stream consequences for patient care, laboratory operations, and total costs. METHODS: The final workflow included rapid enzymatic hydrolysis, rapid liquid chromatographic methods, and time-of-flight mass spectrometry for detection. In total, 84 drugs and metabolites were included and reported qualitatively using 11 isotopically labeled internal standards selected to represent compound classes, retention time, and expected abundances to control for method inefficiencies and matrix suppression/enhancement. The method performance validation included 420 individual urine specimens. RESULTS: Of the 420 samples screened by immunoassay, 117 failed to confirm by mass spectrometry and were immunoassay false positives. None of these 117 samples screened positive on the liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS) assay. The LC-TOF-MS method failed to detect 1 sample in each of the following classes: buprenorphine, ethanol markers, and opiates owing to concentrations below the established cutoffs. Out of 579 samples, 275 (47.4%) screened positive by LC-TOF-MS for nicotine and at least 2 of its metabolites. Quantitative creatinine comparison to an existing Jaffe method yielded a slope of 0.91 and a correlation coefficient of 0.96. CONCLUSIONS: We investigated whether immunoassay-based drug screening and creatinine quantification could be sufficiently replaced by a rapid LC-TOF-MS screen with higher specificity and accuracy than existing methods. The LC-LC-TOF-MS method is a sensitive and more specific way to screen for drugs, providing creatinine quantification and potential novel specimen validity testing with the inclusion of nicotine metabolites.

Up in Smoke: Uncovering a Lack of Evidence for Proton Pump Inhibitors as a Source of Tetrahydrocannabinol Immunoassay False Positives.

Objective: It is recommended that positives in immunoassay drug screens be followed up with more specific confirmatory testing. The drug package insert for pantoprazole mentions reports of false-positive urine screening tests for tetrahydrocannabinol in patients receiving proton pump inhibitors, but no method details or data are given, referenced, or found in literature searches. Thus, we investigated this using our laboratory's assay. Methods: A spiked sample and samples from 32 patients taking a proton pump inhibitor were analyzed using the EMIT II Plus Cannabinoid assay with a 20 ng/mL cutoff. Additionally, we examined urine samples from 50 patients with false-positive or low-positive screens for evidence of a proton pump inhibitor. To determine whether O-desmethyl pantoprazole sulfate, the major metabolite, shares any structural or electrostatic similarity to suggest a basis for cross-reactivity in the immunoassay, we used computational techniques for analyses. Molecular electrostatic potential energy (MEP) maps were calculated for the global minimum conformers, and the maximum common substructure Tanimoto similarity was calculated for the modeled compounds. Results: Neither the spiked sample nor the patient samples were found to screen positive. None of the false-positive or low-positive screens were found to contain a proton pump inhibitor. Computational studies showed very little similarity in shape or electrostatics between the two molecules. Conclusions: We find no supporting evidence of pantoprazole as the cause of false positives in the EMIT II Plus Cannabinoid assay and caution the use of proton pump inhibitors as an explanation for tetrahydrocannabinol immunoassay false positives.

Parathyroid hormone related protein concentration in human serum and CSF correlates with age.

BACKGROUND: Parathyroid Hormone-Related Protein (PTHrP) is involved in intracellular calcium (Ca) regulation, and has been demonstrated to participate in regulation of Ca in brain cells, activation of neurons, and modulation of pain. However, there are conflicting reports regarding the presence of PTHrP in CSF. DESIGN AND METHODS: PTHrP and Ca were quantified in paired CSF and serum samples using mass spectrometry-based methods. Associations between PTHrP and Ca concentrations with age, sex and concentrations of nine CSF diagnostic markers in a set of 140 paired serum and CSF patient samples were evaluated. RESULTS: The observed median PTHrP concentration in CSF was 51 times higher than in serum; the median concentration of Ca in CSF was 1.8 times lower than in serum. We observed positive correlation between concentrations of PTHrP in CSF and serum (p=0.013). Distribution of PTHrP concentrations in serum was associated with age (p=0.0068) and the concentrations were higher in women. In samples with serum calcium concentrations within the reference intervals (n=118), central 95% distribution of concentrations for Ca-CSF, PTHrP-serum and PTHrP-CSF were 5.4 (4.5-6.1) mg/dL, 1.2 (0.5-2.5) pmol/L, 62 (22-125) pmol/L, respectively. CONCLUSIONS: Our data demonstrate that PTHrP is a normal constituent of human CSF with median concentrations 51 fold higher than in serum. Elevated serum PTHrP concentrations were positively correlated with age and significantly higher in women. Our data suggest that CSF could be a significant source of circulating PTHrP.