Urinary Concentrations Research Articles

Presence of N, N'-Substituted p-Phenylenediamine-Derived Quinones in Human Urine.

Human exposure to various N,N'-substituted p-phenylenediamine-derived quinones (PPDQs) has been of increasing concern. Recent studies have examined N-phenyl-N'-(1,3-dimethylbutyl)-p-phenylenediamine-derived quinone (6PPDQ) in human urine to evaluate human exposure. However, other PPDQs in human urine have not been thoroughly investigated. This study analyzed six PPDQs in urine collected from 149 healthy individuals in Taizhou, China. All target PPDQs were detected, with 6PPDQ (mean 2.4 ng/mL, <limit of detection (LOD)-19 ng/mL) and 2-(cyclohexylamino)-5-(phenylamino)cyclohexa-2,5-diene-1,4-dione (CPPDQ; 2.1 ng/mL, <LOD-24 ng/mL) being the most prevalent. Human urinary concentrations of 2,5-bis((5-methylhexan-2-yl)amino)cyclohexa-2,5-diene-1,4-dione (77PDQ; mean 1.5 vs. 0.87 ng/mL; p = 0.013) and 2,5-bis(o-tolylamino)cyclohexa-2,5-diene-1,4-dione (mean 1.1 vs. 0.62 ng/mL; p = 0.027) were significantly higher in females compared to males. For CPPDQ (p < 0.01) and 6PPDQ (p < 0.01), a decrease was observed in urinary concentrations as participants aged. The daily excretion (DE) of PPDQs through urine was estimated for Chinese adults. The highest average DE was recorded for 6PPDQ at 81 ng/kg-bw/day, with a range from <0.5 to 475 ng/kg-bw/day. Following this, CPPDQ had a mean DE of 68 ng/kg-bw/day (range <0.5-516 ng/kg-bw/day), and 77PDQ had a mean DE of 30 ng/kg-bw/day (<0.5-481 ng/kg-bw/day). This study is the first to explore the presence of various PPDQs in human urinary samples, which is essential for assessing the potential health risks associated with these substances.

Urine is better for rare earth elements bimonitoring in long-term exposed population: An exposure-response relationship study

With the soaring use of rare earth elements (REEs) worldwidely in high-technology and clean energy industries, there were growing concerns for adverse health effect from the REEs exposure. However, there is a lack of biomonitoring research concerning both urine and blood in population with definite exposure. We performed a biomonitoring study that involved 103 REEs exposed males and 110 males as non-REEs exposed controls. We measured the levels of REEs in environment and urine and blood samples from participants, and explored the exposure-response relationship between REEs in environment and body fluids. The effects of exposure duration and smoking status on the internal exposure level of REEs were also investigated. The results showed environmental REEs level of exposure group was significantly higher than that of control group (range of geometric mean of exposure vs. control: 1.08-4.07 × 104 ng/m3 vs. <LOD-2.16 × 102 ng/m3). Six elements with detection rates higher than 60% in blood or urine samples were lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), gadolinium (Gd). We found the REEs concentrations both in urine and blood of exposure population were significantly higher than controls, median range of the above 6 elements of urine and blood was 0.02–1.06 μmol/mol vs. <LOD-0.01 μmol/mol creatinine and 0.01–0.79 μg/L vs. <LOD-0.38 μg/L. The correlations between both blood and urine level of REEs and environment level showed significant. The correlation coefficients with urine levels are higher than with blood. Biomonitoring results showed good exposure-response relationship in urine REEs, while no positive response in blood samples. Smoking status, drinking status and years of exposure showed little effect on the level of REEs. Our results suggested that both blood and urine can be used to monitor REEs exposure, while urinary REEs is promising for risk assessment in population.

Investigating thyroid function and iodine status in adolescents with and without paediatric major depressive disorder.

Depression has been associated with subclinical hypothyroidism and altered hypothalamic-pituitary-thyroid axis functioning. Adequate iodine nutrition is essential for healthy thyroid functioning. We therefore determined associations of iodine and thyroid status with paediatric major depressive disorder (pMDD) among Swiss adolescents and explored whether associations are sex-specific and mediated by stress. We conducted a matched case-control study in 95 adolescents with diagnosed pMDD and 95 healthy controls. We assessed depression severity using the Children's Depression Rating Scale-Revised and stress using the perceived stress scale (PSS) and measuring hair cortisol levels. We determined iodine status by measuring urinary iodine concentrations (UIC) and thyroid status by thyroid-stimulating hormone (TSH) and free thyroxine (FT4) in serum. Median (IQR) UIC did not differ between cases (121 (87, 174) µg/l) and controls (114 (66, 183) μg/l, P = 0·3). Median TSH and FT4 were lower in cases than controls (TSH: 1·36 (0·91, 2·00) mlU/l v. 1·50 (1·18, 2·06) mlU/l, P = 0·039; FT4: 14·7 (12·9, 16·9) pmol/l v. 15·7 (14·3, 17·2) pmol/l, P = 0·004). The prevalence of hypothyroxinaemia (normal TSH; low FT4) was higher among female cases than controls (21 % v. 4%, P = 0·006). PSS scores were higher while hair cortisol was lower in cases than controls (PSS: 25 (20, 28) v. 11 (7, 15), P < 0·001; cortisol: 2·50 (1·34, 3·57) pg/mg v. 3·23 (1·79, 4·43) pg/mg, P = 0·044). After adjusting for confounders, the associations of TSH and hair cortisol with pMDD were no longer significant. Furthermore, TSH and FT4 were not associated with PSS scores and hair cortisol levels. Summarising, iodine nutrition was adequate for adolescents with and without pMDD. However, FT4 concentrations were lower in those with pMDD, and 1 in 5 female adolescents with pMDD were hypothyroxinaemic.

Effect of Adopting a Gluten-Free Diet on Exposure to Arsenic and Other Heavy Metals in Children With Celiac Disease: A Prospective Cohort Study.

Lifelong adherence to a gluten-free diet (GFD) is the primary treatment of celiac disease (CeD), a gluten-driven enteropathy. Concerns have been raised about increased exposure to arsenic from a GFD because rice, which naturally bioaccumulates arsenic, is commonly used as a substitute for gluten-containing grains such as wheat. We hypothesize that arsenic exposure increases in newly diagnosed children with CeD after they adopt a GFD. This is a single-center prospective longitudinal cohort study of children (age 2-18 years) with elevated celiac serology who underwent a diagnostic endoscopy before initiation of a GFD between January and May 2022. The primary outcome was change in urinary arsenic concentration between endoscopy and after 6 months on a GFD. Of the 67 recruited participants, 50 had a biopsy diagnostic of CeD and were invited to continue the study. Thirty-five participants completed sample collection. Participants were from a middle-class, well-educated population that was predominantly White with presenting symptoms of abdominal pain (51%) and diarrhea (29%). After 6 months on a GFD, there was a significant increase in the median urinary arsenic concentration (3.3 µg/L vs 13.6 µg/L, P = 0.000004). In regression models, family history of CeD and Hispanic ethnicity were associated with having a higher urinary arsenic concentration after 6 months on a GFD. Children with newly diagnosed CeD have increased arsenic exposure shortly after transitioning to a GFD. While the arsenic levels were well below acutely toxic concentrations, the clinical impact of chronic exposure to mildly elevated arsenic levels is unknown.

Impaired 11β-hydroxysteroid dehydrogenase type 2 activity in kidney disease disrupts 11-oxygenated androgen biosynthesis.

11-oxygenated androgens are a group of adrenal-derived steroids that require peripheral activation. In vitro data highlight a putative role for 11β-hydroxysteroid dehydrogenase type 2 (HSD11B2) in 11-oxygenated androgen biosynthesis, converting 11β-hydroxyandrostenedione (11OHA4) to 11-ketoandrostenedione (11KA4), the direct precursor of the potent androgen 11-ketotestosterone (11KT). As the kidney is the major site of HSD11B2 expression, we hypothesized that patients with chronic kidney disease (CKD) would have reduced 11-oxygenated androgen biosynthesis due to impaired HSD11B2 activity. To determine the role of HSD11B2 in 11-oxygenated androgen biosynthesis using a human CKD cohort alongside complementary cell culture and computational modeling approaches. Cross-sectional observational study of patients with CKD (n=85) and healthy controls (n=46) measuring serum and urinary concentrations of glucocorticoids, classic and 11-oxygenated androgens by liquid chromatography-tandem mass spectrometry. A computational model of peripheral 11-oxygenated androgen biosynthesis was fitted to the serum data to calculate relative HSD11B2 expression levels for each participant. HSD11B2 activity declined with eGFR, evidenced by higher cortisol (F)/cortisone (E) ratios in CKD patients compared to controls (p<0.0001). Serum concentrations of E, 11KA4, 11KT and 11β-hydroxytestosterone were lower in patients with CKD compared to controls (p<0.0001 for each). A computational model based on enzyme kinetic parameters of HSD11B2, 11β-hydroxysteroid dehydrogenase type 1, 17β-hydroxysteroid dehydrogenase type 2 and aldo-keto reductase 1C3 confirmed HSD11B2 as the key enzyme responsible for reduced 11-oxygenated androgen biosynthesis in CKD. Predicted HSD11B2 expression correlated with eGFR. This is the first in vivo study to confirm a central role for renal HSD11B2 in 11-oxygenated androgen biosynthesis. Determining the clinical implications of this observation for patients with CKD requires further research.

Development of a Generic Physiologically Based Kinetic Model for the Prediction of Internal Exposure to Organophosphate Pesticides.

Since their introduction into agriculture, the toxicity of organophosphate (OP) pesticides has been widely studied in animal models. However, next generation risk assessment (NGRA) intends to maximize the use of novel approach methodologies based on in vitro and in silico methods. Therefore, this study describes the development and evaluation of a generic physiologically based kinetic (PBK) model for acute exposure to OP pesticides in rats and humans using quantitative structure property relationships and data from published in vitro studies. The models were evaluated using in vivo studies from the literature for chlorpyrifos, diazinon, fenitrothion, methyl-parathion, ethyl-parathion, dimethoate, chlorfenvinphos, and profenofos. Evaluation was performed by comparing simulated and in vivo observed time profiles for blood, plasma, or urinary concentrations and other toxicokinetic parameters. Of simulated concentration-time profiles, 87 and 91% were within a 5-fold difference from observed toxicokinetic data from rat and human studies, respectively. Only for dimethyl-organophosphates further refinement of the model is required. It is concluded that the developed generic PBK model provides a new tool to assess species differences in rat and human kinetics of OP pesticides. This approach provides a means to perform NGRA for these compounds and could also be adopted for other classes of compounds.

Sociodemographic and dietary determinants of glyphosate exposure in a NYC-based pregnancy cohort

Previous studies have provided evidence for associations between glyphosate and aminomethylphosphonic acid (AMPA) exposure and adverse birth outcomes. However, few pregnancy cohort studies have investigated dietary and other determinants of glyphosate and AMPA exposure. We aimed to identify dietary and sociodemographic factors that predict glyphosate and AMPA exposure in a contemporary, urban pregnancy cohort in the US. The study included 725 pregnant participants from the New York University Children's Health and Environment Study (NYU CHES) in New York City. Urinary concentrations of glyphosate and AMPA, determined by high-performance liquid chromatography and tandem mass spectrometry, were analyzed in urine collected from NYU CHES participants across three prenatal time points. The Diet Health Questionnaire II was completed to capture dietary intake during the prenatal period. Descriptive statistics and bivariate linear models were used to assess determinants of urinary glyphosate and AMPA concentrations. Median urinary glyphosate and AMPA levels were 0.36 ng/mL and 0.37 ng/mL, respectively. Lower glyphosate levels were associated with younger age, obesity, public insurance, being single, and lower educational attainment. Nuts, seeds and whole grain intake was associated with increased urinary glyphosate concentrations. Urinary glyphosate concentrations were lower in summer than in winter. The study findings highlight widespread exposure to glyphosate and AMPA in this pregnancy cohort, with nuts/seeds and whole grains identified as possible dietary sources of exposure. High detection rates in the study population necessitate further research on dietary exposure patterns and perinatal outcomes to inform targeted interventions and reduce exposure in vulnerable populations.

8046 Milk Provision as a Potential Intervention to Improve Iodine Status in a Pregnant Cohort in Northern Ireland (NI), UK: A Randomised Controlled Trial

Abstract Disclosure: L.R. Kayes: None. K.R. Mullan: None. J.V. Woodside: None. Iodine deficiency is the leading cause of reversible mental retardation worldwide. During pregnancy there is ∼50% increase in iodine requirement for foetal neurodevelopment. The most recently studied cohorts of pregnant women across Ireland and UK consistently demonstrated mild iodine deficiency (target median urinary iodine concentration (mUIC) ≥150 µg/L). A recent study in NI has shown that women of child-bearing age provided with three litres of cow’s milk per week had improved iodine status. We wished to explore whether a similar milk intervention might improve iodine status during pregnancy. Pregnant women were recruited at their first antenatal appointment, and all were provided with the British Dietetic Association’s iodine food fact sheet. Participants were randomised to either milk intervention, (four litres/week milk delivered weekly free of charge), or no milk delivery (control group). Patients were stratified based on use of an iodine-containing multivitamin prior to randomisation. Urine was collected at recruitment, after 12-week intervention period, in the third trimester and 6-12 weeks postnatally along with an infant urine sample when possible. A total of 55 urine samples were collected from infants. A total of 118 women were recruited. Participants had a mean age of 32 years, (range 21 - 45) with a mean BMI of 27.5 kg/m2. Mean gestational age was 12 weeks (IQR 12-13 weeks). The mUIC in the whole group women was 53.2 µg/L in the first trimester. In the control group the mUIC at the first study visit (n=59) was 93.4 µg/L (22% achieving iodine sufficiency); at the second visit (n=38), the mUIC was 158.7 µg/L with 34% achieving sufficiency. In the milk group the mUIC at the first study visit (n=59) was 82.0 µg/L with 10% achieving sufficiency (no statistical significance between groups, p=0.08); at the second visit (n=43), mUIC was 154.7 µg/L with 37% achieving sufficiency (no statistical difference between groups p=0.78). The mean change in UIC between the first and second trimesters was 44.5 and 88.1 µg/L in the control and milk arms respectively (p=0.27). In this cohort, 71.4% of women reported taking an iodine-containing multivitamin in the first trimester of pregnancy with no difference between group randomised to milk intervention (p=0.95). In the infants, mUIC was 124.1 µg/L with no statistically difference in the proportion reaching iodine sufficiency according to maternal randomisation (p=0.64) or maternal use of an iodine-containing multivitamin (p=0.95). In this cohort, there was no significant improvement in mUIC in those receiving a milk intervention. Presentation: 6/1/2024

8046 Milk Provision as a Potential Intervention to Improve Iodine Status in a Pregnant Cohort in Northern Ireland (NI), UK: A Randomised Controlled Trial

Abstract Disclosure: L.R. Kayes: None. K.R. Mullan: None. J.V. Woodside: None. Iodine deficiency is the leading cause of reversible mental retardation worldwide. During pregnancy there is ∼50% increase in iodine requirement for foetal neurodevelopment. The most recently studied cohorts of pregnant women across Ireland and UK consistently demonstrated mild iodine deficiency (target median urinary iodine concentration (mUIC) ≥150 µg/L). A recent study in NI has shown that women of child-bearing age provided with three litres of cow’s milk per week had improved iodine status. We wished to explore whether a similar milk intervention might improve iodine status during pregnancy. Pregnant women were recruited at their first antenatal appointment, and all were provided with the British Dietetic Association’s iodine food fact sheet. Participants were randomised to either milk intervention, (four litres/week milk delivered weekly free of charge), or no milk delivery (control group). Patients were stratified based on use of an iodine-containing multivitamin prior to randomisation. Urine was collected at recruitment, after 12-week intervention period, in the third trimester and 6-12 weeks postnatally along with an infant urine sample when possible. A total of 55 urine samples were collected from infants. A total of 118 women were recruited. Participants had a mean age of 32 years, (range 21 - 45) with a mean BMI of 27.5 kg/m2. Mean gestational age was 12 weeks (IQR 12-13 weeks). The mUIC in the whole group women was 53.2 µg/L in the first trimester. In the control group the mUIC at the first study visit (n=59) was 93.4 µg/L (22% achieving iodine sufficiency); at the second visit (n=38), the mUIC was 158.7 µg/L with 34% achieving sufficiency. In the milk group the mUIC at the first study visit (n=59) was 82.0 µg/L with 10% achieving sufficiency (no statistical significance between groups, p=0.08); at the second visit (n=43), mUIC was 154.7 µg/L with 37% achieving sufficiency (no statistical difference between groups p=0.78). The mean change in UIC between the first and second trimesters was 44.5 and 88.1 µg/L in the control and milk arms respectively (p=0.27). In this cohort, 71.4% of women reported taking an iodine-containing multivitamin in the first trimester of pregnancy with no difference between group randomised to milk intervention (p=0.95). In the infants, mUIC was 124.1 µg/L with no statistically difference in the proportion reaching iodine sufficiency according to maternal randomisation (p=0.64) or maternal use of an iodine-containing multivitamin (p=0.95). In this cohort, there was no significant improvement in mUIC in those receiving a milk intervention. Presentation: 6/1/2024

6621 Iodine Nutritional Status in Different Trimesters of Pregnancy: A Prospective Observational Study in Taiwan

Abstract Disclosure: L. Pan: None. C. Yeh: None. C. Yang: None. C. Hwu: None. H. Chen: None. F. Wang: None. C. Huang: None. Pregnant women are susceptible to iodine deficiency. Iodine nutritional studies in pregnant women were mostly cross sectional evaluations of iodine status. This prospective observational study was designed to longitudinally follow the iodine status of pregnant women in different trimesters of pregnancy and to assess the clinical parameters and dietary influences on iodine status. A total of 209 pregnant women in their first trimester were enrolled in Taipei Veterans General Hospital, Taiwan from March 2019 to March 2023. Subjects provided a spot urine sample and completed a food frequency questionnaire at each trimester. Urinary iodine concentration (UIC) was measured by inductively coupled plasma mass spectrometry. The median UIC for the first, second and third trimester was 156.3 μg/L (Interquartile range [IQR]: 100.7-241.1), 146.2 μg/L (84.6-224.4), and 169.9 μg/L (94.4-283.5), respectively. Mild iodine deficiency was observed in the second trimester despite adequate iodine status in the first and third trimesters. Compared to the first trimester, intake of seaweed (p = 0.004, p &amp;lt; 0.001, respectively), fish (p &amp;lt; 0.001, p &amp;lt; 0.001, respectively), and prenatal multivitamins (p &amp;lt; 0.001, p &amp;lt; 0.001, respectively) increased in the second and third trimesters. A gradual increment in dairy product intake was also noted between each trimesters through pregnancy (p &amp;lt; 0.001, p = 0.004, respectively). Multivariate analysis indicated a positive association between multiparity and increased UIC in the second trimesters (multiparity vs. uniparity; adjusted odds ratio [OR]: 2.273, 95% confidence interval [CI]: 1.202-4.310). Additionally, increased prenatal multivitamin intake elevated the odds of adequate UIC in the third trimester (≥ 3 days/week vs. ≤ 1 day/week; adjusted OR: 2.890, 95% CI: 1.351-6.173). This study revealed the longitudinal changes in iodine status during pregnancy and suggested that women who regularly took prenatal multivitamins and were multiparous were more likely to have adequate iodine nutrition. Presentation: 6/2/2024

7899 Mixture Of Urinary Concentrations Of Phenols And Phthalate Metabolites In Relation To Serum Levels Of Lipid Biomarkers Among Pregnant Women: Results From The EARTH Study

Abstract Disclosure: X. Shen: None. M. Génard-Walton: None. P.L. Williams: None. T. James-Todd: None. J.B. Ford: None. K.M. Rexrode: None. D. Zhang: None. J.E. Chavarro: None. R.B. Hauser: None. L. Minguez Alarcon: None. Given the limited evidence on endocrine-disrupting chemicals (EDCs), evaluated as mixture, in relation to lipid biomarkers during pregnancy, we examined whether mixtures of urinary concentrations of bisphenol A (BPA), parabens and phthalate metabolites were associated with serum lipid levels among 175 pregnant women who enrolled in Environment and Reproductive Health (EARTH) Study from 2005 to 2017. The outcomes were assessed at one random timepoint pre women during pregnancy, including serum levels of total triglycerides, total cholesterol, high-density lipoprotein (HDL), non-HDL, and low-density lipoprotein (LDL). We grouped the chemical biomarkers [di(2-ethylhexyl) phthalate (DEHP) metabolites and BPA; other phthalate metabolites; parabens] assessed in urine collected at the same time and applied Bayesian Kernel Machine Regression (BKMR) and quantile g-computation to evaluate the associations while adjusting for confounders [urine specific gravity, age, pre-pregnancy body mass index (BMI), race, education level, infertility diagnosis, mode of conception, number of babies in this pregnancy and trimester]. In the adjusted BKMR models, we found no associations between the overall chemical biomarker mixture and lipid levels for example total cholesterol [mean difference (95% CRI, credible interval) = 0.02 (-0.31, 0.34)] and LDL [mean difference (95% CRI) = 0.10 (-0.22, 0.43)] when comparing concentrations at the 75th to the 25th percentile. When stratified by BMI, we found suggestive positive relationships between the paraben group and total cholesterol and LDL among women with high BMI [mean difference (95% CRI) = 0.16 (-0.23, 0.55) and 0.16 (-0.25, 0.57)], but not among women with low BMI [mean difference (95% CRI) = -0.02 (-0.16, 0.12) and -0.02 (-0.15, 0.12)]. Among the paraben group, propylparaben was driving the associations among high BMI group [mean difference (95% CRI) = 0.25 (-0.26, 0.75) and 0.35 (-0.25, 0.95)], but not among low BMI group [mean difference (95% CRI) = 0.00 (-0.06, 0.07) and 0.00 (-0.07, 0.07)]. In addition, we found suggestive positive associations of DEHP metabolites and BPA group with total cholesterol, non-HDL and LDL among women with lower BMI, but none of the chemicals was identified as an important contributor to these associations. This study highlights that the associations between EDCs and cardiovascular biomarkers, such as circulating lipids, differ by BMI among pregnant women. Presentation: 6/1/2024

Sociodemographic and dietary predictors of maternal and placental mycoestrogen concentrations in a US pregnancy cohort.

Zearalenone (ZEN) is a mycotoxin contaminating grains and processed foods. ZEN alters nuclear estrogen receptor α/β signaling earning its designation as a mycoestrogen. Experimental evidence demonstrates that mycoestrogen exposure during pregnancy is associated with altered maternal sex steroid hormones, changes in placental size, and decreases in fetal weight and length. While mycoestrogens have been detected in human biospecimens worldwide, exposure assessment of ZEN in US populations, particularly during pregnancy, is lacking. To characterize urinary and placental concentrations of ZEN and its metabolites in healthy US pregnant people and examine demographic, perinatal, and dietary predictors of exposure. Urine samples were collected in each trimester from pregnant participants in the UPSIDE study and placenta samples were collected at delivery (Rochester, NY, n = 317). We used high performance liquid chromatography and high-resolution tandem mass spectrometry to measure total urinary (ng/ml) and placental mycoestrogens (ng/g). Using linear regression and linear mixed effect models, we examined associations between mycoestrogen concentrations and demographic, perinatal, and dietary factors (Healthy Eating Index [HEI], ultra-processed food [UPF] consumption). Mycoestrogens were detected in 97% of urines (median 0.323 ng/ml) and 84% of placentas (median 0.012 ng/g). Stability of urinary mycoestrogens across pregnancy was low (ICC: 0.16-0.22) and did not correlate with placental levels. In adjusted models, parity (multiparous) and pre-pregnancy BMI (higher) predicted higher urinary concentrations. Birth season (fall) corresponded with higher placental mycoestrogens. Dietary analyses indicated that higher HEI (healthier diets) predicted lower exposure (e.g., Σmycoestrogens %∆ -2.03; 95%CI -3.23, -0.81) and higher percent calories from UPF predicted higher exposure (e.g., Σmycoestrogens %∆ 1.26; 95%CI 0.29, 2.24). The mycotoxin, zearalenone (ZEN), has been linked to adverse health and reproductive impacts in animal models and livestock. Despite evidence of widespread human exposure, relatively little is known about predictors of exposure. In a pregnant population, we observed that maternal ZEN concentrations varied by maternal pre-pregnancy BMI and parity. Consumption of ultra-processed foods, added sugars, and refined grains were linked to higher ZEN concentrations while healthier diets were associated with lower levels. Our research suggests disparities in exposure that are likely due to diet. Further research is needed to understand the impacts of ZEN on maternal and offspring health.

Disruption of mitochondrial electron transport impairs urinary concentration via AMPK-dependent suppression of aquaporin-2.

Urinary concentration is an energy-dependent process that minimizes body water loss by increasing aquaporin-2 (AQP2) expression in collecting duct (CD) principal cells. To investigate the role of mitochondrial (mt) ATP production in renal water clearance, we disrupted mt electron transport in CD cells by targeting ubiquinone (Q) binding protein QPC (UQCRQ), a subunit of mt complex III essential for oxidative phosphorylation. QPC-deficient mice produced less concentrated urine than controls, both at baseline and after type 2 vasopressin receptor stimulation with desmopressin. Impaired urinary concentration in QPC-deficient mice was associated with reduced total AQP2 protein levels in CD tubules, while AQP2 phosphorylation and membrane trafficking remained unaffected. In cultured inner medullary CD cells treated with mt complex III inhibitor antimycin A, the reduction in AQP2 abundance was associated with activation of 5' adenosine monophosphate-activated protein kinase (AMPK) and was reversed by treatment with AMPK inhibitor SBI-0206965. In summary, our studies demonstrated that the physiological regulation of AQP2 abundance in principal CD cells was dependent on mt electron transport. Furthermore, our data suggested that oxidative phosphorylation in CD cells was dispensable for maintaining water homeostasis under baseline conditions, but necessary for maximal stimulation of AQP2 expression and urinary concentration.

B-057 Evaluating the Use of Unconcentrated Urine for Protein Electrophoresis and Immunofixation: An Alternative Approach for Efficient Analysis

Abstract Background Protein electrophoresis is a widely employed technique for separating and analyzing proteins based on their charge and size. Traditionally, concentrated urine samples have been utilized to enhance protein detection sensitivity. However, this study explores the feasibility of using unconcentrated urine as an alternative sample type for protein electrophoresis (UEL) and immunofixation (UIF) to streamline the analysis process and reduce costs and turnaround time in clinical laboratories. It offers a more efficient and resource-friendly alternative. Methods Both UEL and UIF were performed on routinely ordered 80 urine specimens (random or 24 Hr.) for patient management and performed by both concentrated (X40) by Helena and unconcentrated using Sebia (HYDRAGEL 9IF gels using HYDRAGEL BJ dynamic mask procedure. The electrophoretic patterns obtained from unconcentrated urine were compared with those from concentrated urine. Various electrophoretic parameters, including resolution, sensitivity, and reproducibility, were evaluated to determine the reliability of unconcentrated urine in protein electrophoresis. In addition, retrospective analysis was performed before and after implementation on all urine specimens ordered to analyze if there were any differences in the monoclonal band distributions in the population. Results All the protein concentrations were correlated well with 95-100% agreement between the methods (Figure 1) clear separation of protein fractions. Serial dilution studies of monoclonal band analyses showed that these bands can be detected at 1.25 mg/dL using an unconcentrated method. Retrospective analysis of patient data before and after the implementation of the new method shows no differences in the identification of monoclonal proteins (Helena [69 of 872 total patients tested (8.0%)] vs. Sebia [90/800 (11.0%)]. Conclusions These results indicate that unconcentrated urine can be successfully utilized for UEL and UIF with comparable outcomes to traditional concentrated samples. Utilizing unconcentrated urine simplifies the sample preparation process and presents a promising alternative.

B-173 Cystine Analysis in Urine Sample Using the Laser Diode Thermal Desorption technique “LDTD”/Mass Spectrometry (Luxon)

Abstract Background Cystine is a polar amino acid found in the urine of healthy individuals; however, a genetic defect in renal transport can lead to cystine crystals and stones. A usual laboratory approach for diagnosing and monitoring cystinuria is to measure cystine concentration in 24-hour urine. The disease is rare, affecting around 1 in every 10,000 live births. The objective of this study was to develop a rapid assay using Tandem Mass Spectrometry methodology with an internal standard of Cystine derivatized in a urine sample, Luxon Ion Source®, Laser Diode Thermal Desorption (LDTD) technology. Methods The Luxon is a sample introduction and ionization source with a coupled laser diode, providing thermal uniformity, precision, accuracy and speed. Thermally desorbed neutral molecules are analyzed by mass spectrometry (MS). Initially, the sample was derivatized to make the cystine thermally vaporize, followed by liquid-liquid extraction assisted by salting out (SALLE). The isotope-labeled internal standard (Cystine-d4), homemade calibration curve and water quality controls were used for quantification. The analysis was performed using a Sciex 6500+ Triple Quad in APCI (Atmospheric Pressure Chemical Ionization) positive mode. The ratio of peak area to internal standard (ISTD) was used to normalize the signal. A linear regression model with 1/X2 weighting was used to obtain the calibration curve equation and determine the correlation coefficient (r2). Parallelism was assessed by comparing the (r2) of calibration curves prepared in water with six curves prepared with biological matrices. Intra- and interassay precision and accuracy were determined by measuring control samples ranging from low to high concentration in five replicates over 3 consecutive days. For urine analysis, aqueous calibration curves in the concentration range of 1 to 100 mg/L (n=3) showed excellent linearity. The following parameters were evaluated for quantitative analysis: linearity, precision, accuracy, carryover and parallelism.​ Results The calibration curve resulted in a linear response across the entire concentration range, the correlation coefficient (r) was 0.99. For each quality control concentration, CVs were &amp;lt;10%, meeting desirable imprecision and precision specifications. The intraassay CVs were 1.0-2.56% and the interassay CVs were 2.15-4.58% for precision, which reflects the agreement between repeated measurements. Precision results showed similar results, intra-assay CVs were 2.23-3.66% and inter-assay CVs were 1.0-2.77% Conclusions The new LDTD-MS/MS method for the determination of urinary Cystine was accepted after verification and validation of quality control parameters. The technique proved to be effective for reliably and quickly quantifying cystine in urinary samples from human patients, being a valuable tool in the diagnosis and prevention of cystine kidney stones. The assay developed, using the Luxon Ion Source®, based on LDTD (Laser Diode Thermal Desorption) technology, proved to be ultrafast for measuring derivatized cystine in urine

Characterization of pesticide exposures and their associations with asthma morbidity in a predominantly low-income urban pediatric cohort in Baltimore City

BackgroundPesticides may impact respiratory health, yet evidence of their impact on pediatric asthma morbidity is limited, particularly among urban children. ObjectiveTo characterize pesticide biomarker concentrations and evaluate their associations with pediatric asthma morbidity among predominantly low-income, Black children in Baltimore City, USA. MethodsWe measured urinary concentrations of 10 biomarkers for pyrethroid insecticides (cyfluthrin:4F-3PBA, permethrin:3PBA), organophosphate insecticides (chlorpyrifos:TCPY, malathion:MDA, parathion:PNP, diazinon:IMPY), and herbicides (glyphosate:AMPA, GPS; 2,4-dicholorphenoxyacetic acid:2,4-D; 2,4,5-tricholorphenoxyacetic acid:2,4,5-T) among 148 children (5–17 years) with established asthma. Urine samples and asthma morbidity measures (asthma symptoms, healthcare utilization, lung function and inflammation) were collected every three months for a year. Generalized estimating equations were used to examine associations between pesticide biomarker concentrations and asthma morbidity measures, controlling for age, sex, race, caregiver education, season, and environmental tobacco smoke. In sensitivity analyses, we assessed the robustness of our results after accounting for environmental co-exposures. ResultsFrequently detected (≥90% detection) pesticide biomarker concentrations (IMPY, 3PBA, PNP, TCPY, AMPA, GPS) varied considerably within children over the follow-up period (intraclass correlation coefficients: 0.1–0.2). Consistent positive significant associations were observed between the chlorpyrifos biomarker, TCPY, and asthma symptoms. Urinary concentrations of TCPY were associated with increased odds of coughing, wheezing, or chest tightness (adjusted Odds Ratio, aOR, TCPY:1.60, 95% Confidence Interval, CI:1.17–2.18). Urinary concentrations of TCPY were also associated with maximal symptom days (aOR:1.38, CI:1.02–1.86), exercise-related symptoms (aOR:1.63, CI:1.09–2.44), and hospitalizations for asthma (aOR:2.84, CI:1.08–7.43). We did not observe consistent evidence of associations between the pesticide exposures assessed and lung function or inflammation measures. ConclusionAmong predominantly Black children with asthma, we found evidence that chlorpyrifos is associated with asthma morbidity. Further research is needed to assess the contribution of pesticide exposures to pediatric respiratory health and characterize exposure sources among vulnerable populations to inform targeted interventions against potentially harmful pesticide exposures.

B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis

Abstract Background Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities. Methods The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (&amp;gt;0.06ng/g Creatinine). Results Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney. Conclusions The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.

Determination of multiple analytes in urine using label-free SERS coupled with simple sample pretreatments

BackgroundA key restriction of label-free surface-enhanced Raman spectroscopy (SERS) in analysis of objects with complex composition (including with several target analytes) is the competition of mixture components for interaction with SERS-active surface. This leads to poor selectivity of the analysis of such mixtures (e.g., body fluids) and the need to use advanced sample pretreatment procedures such as HPLC or TLC. Therefore, this work aims to develop a set of simple and fast pretreatment steps (dilution, pH correction, etc.) to increase the sorption of the target analyte, reduce the sorption of admixtures, and prevent suppression of the target analyte SERS signal. ResultsWe have developed label-free SERS assay suitable for the determination of three analytes (methotrexate, cephalosporin antibiotic, and creatinine) in one real urine sample as a model matrix with complex and deviating composition. The choice of drugs is justified by the need to monitor their concentration in urine during joint drug treatment of cancer patients with concomitant bacterial infection, while monitoring creatinine concentration helps to evaluate kidney function of the patients. Additionally, three cephalosporin representatives were used in the study to maximize versatility of the assay. As a results, the optimized pretreatment steps enable to eliminate the negative influence of excess of interferences (including other analytes) and achieve precise (≤12% RSD) and accurate (88–111% recovery) determination of several analytes in the therapeutically relevant ranges: 300–3000 μg mL–1 for creatinine, 20–200 μg mL–1 for methotrexate and cephalosporins. SignificanceTherefore, in addition to reporting a new SERS assay for the analysis of body fluids, this study clearly demonstrates the importance of taking into account competitive adsorption processes on the SERS substrate surface. We suggest making this practice mandatory when developing any label-free SERS assay because it enables to maximize the selectivity and accuracy of the analysis as well as to simplify the analysis procedure.

Laccase mimetics as sensing elements for amperometric assay of 5-hydroxyindoleacetic acid in urine

Monitoring of the levels of 5-hydroxyindole-3-acetic acid (5-HIAA) is of significant importance for diagnostics of carcinoid tumors. We propose simple catalytic electrochemical sensors for the determination of 5-HIAA in urine using laccase and its mimetics. Laccase-like nanozymes (LacNZs) were synthesized via a chemical reduction, and resulting PtMn and MnO2 nanoflowers (NFs) demonstrated laccase-like activity similar to the laccase from the Trametes zonata. In addition, these LacNZs showed enhanced stability under a wide range of pH (3.0–7.5), temperatures (4–70 °C), and ionic strengths (up to 500 mM NaCl). The developed PtMn NF/graphite electrode, similar to a laccase/graphite electrode, can detect 5-HIAA with a high sensitivity (25 000 ± 12 A·M−1·m−2 and 1900 ± 9 A·M−1·m−2, respectively) and have linear ranges of 0.3 – 15 μM and 2 – 50 μM. The sensors work at low working potentials with a detection limit of 0.16 and 1.4 μM, covering the normal and pathologic ranges of 5-HIAA (1–50 μM) content in urine. They have been successfully applied to 5-HIAA assay in urine samples of people with various diseases and revealed good recovery values and reproducibility. Additionally, the LacNZ-sensor has the best stability and can be used up to 20 days.

Inflammation and lipoperoxidation in mucopolysaccharidoses type II patients at diagnosis and post-hematopoietic stem cell transplantation

IntroductionMucopolysaccharidosis type II (MPS II) is caused by deficiency of the enzyme iduronate-2-sulfatase; one possible therapy for MPS II is hematopoietic stem cell transplantation (HSCT). It is established that there is excessive production of reactive species in MPS II patients, which can trigger several processes, such as the inflammatory cascade. ObjectivesOur aim was to outline an inflammatory profile and lipoperoxidation of MPS II patients for a better understanding of disease and possible benefits that HSCT can bring in these processes. Materials and MethodsWe investigate oxidative damage to lipids by 15-F2t-isoprostane urinary concentrations and plasma pro-and anti-inflammatory cytokine concentrations in MPS II patients at diagnosis, MPS II post-HSCT patients, and controls. ResultsInterleukin (IL)-1β and IL-17a concentrations were significantly increased and a tendency toward increased IL-6 production in the diagnosis group was verified. We found significant decrease in IL-4 and increase in 15-F2t-isoprostane concentrations in the diagnosis group, while IL-1β, IL-6, IL-17a and 15-F2t-isoprostane concentrations were similar between control and post-HSCT groups. ConclusionsOur study demonstrated that MPS II patients at diagnosis are in a pro-inflammatory state, bringing a novel result showing increased production of IL-17a, an osteoclastogenic cytokine, as well as demonstrating that these patients have oxidative damage to lipids. Furthermore, evidence suggests that HSCT can reduce inflammation and lipoperoxidation in MPS II patients.