Absolute Concentrations Research Articles

Associations of omega-3 fatty acid subclasses with cardiovascular diseases in 500,000 individuals

Abstract Increased blood levels of omega-3 fatty acids have consistently been shown to be associated with reduced risk of cardiovascular diseases (CVD). However, large scale placebo-controlled trials of omega-3 supplementation have had discordant results: in 2018 REDUCE-IT reported a 25% risk reduction, whereas in 2020 STRENGTH reported no risk reduction. The two trials had both different placebos (mineral vs corn oil) and omega-3 formulations (EPA vs DHA+EPA). There is not yet a consensus on what explains these results. Here we describe the profiling of omega-3 fatty acid subclasses, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) with nuclear magnetic resonance (NMR), and test their associations with incident CVD in 500,000 individuals from UK Biobank. We utilized 500 MHz NMR spectrometers to analyze total omega-3 and its subclasses in EDTA plasma samples and employed biomarker-specific quantification algorithms to measure their absolute concentrations. In this study, we developed a novel algorithm to quantify EPA, complementing previously described algorithms for omega-3 and DHA. Correlation (r^2) of this novel NMR-based quantification and gas chromatography-based measurements was 0.77 for EPA. On average, DHA comprised 44.5% of the total omega-3 concentration, EPA accounted for 22.2%, and other subclasses constituted 34.3% in the cohort. We computed age and sex adjusted associations of omega-3, DHA, and EPA with 10-year risk of CVD among individuals who were not taking cholesterol lowering medication. We discovered protective associations: for DHA a hazard ratio of 0.85 (0.86-0.89, 95% confidence interval) per standard deviation versus 0.96 (0.95-0.98) for EPA, and 0.95 (0.93-0.97) for total omega-3 (Figure). Since DHA (used in the STRENGTH trial with a null result) has a significantly stronger epidemiological association than EPA, these results from a large-scale observational study do not support the hypothesis that the differential use of DHA and EPA in the REDUCE-IT and STRENGTH trials explains their divergent results.Figure

Diffuse white matter pathology in multiple sclerosis during treatment with dimethyl fumarate-An observational study of changes in normal-appearing white matter using proton magnetic resonance spectroscopy.

Multiple sclerosis (MS) is an inflammatory demyelinating disease with neurodegenerative features causing risk for neurologic irreversible disability over time. Examination of normal-appearing white matter (NAWM) changes in MS by proton magnetic resonance spectroscopy (1H-MRS), may detect diffuse white matter pathology that is associated with neurodegeneration. In this observational study of in total twenty-six patients with MS, starting treatment with dimethyl fumarate (DMF), we measured the absolute concentration of metabolites in periventricular NAWM using 1H-MRS at baseline and after one and three years of treatment. Metabolite concentrations were analyzed both cross-sectionally, in relation to 10 controls and longitudinally in relation to disease activity. Patients with MS had higher concentrations of myo-inositol (mIns) in NAWM at baseline compared with controls (mean 5.98 ± 1.37 (SD) and 4.32 ± 1.16 (SD), p<0.01, independent samples t-test). The disease duration was inversely correlated with concentrations of total N-acetylaspartate and N-acetylaspartylglutamate (tNA) (r = -0.62, p<0.01) in NAWM as well as positively to the ratio of mIns and tNA (r = 0.51, p = 0.03). Metabolite concentrations during one-year (n = 19) and three-years (n = 11) follow-up were generally stable. The dropouts were caused by treatment switch after one year, mainly due to new MRI activity. Cross-sectional analyses showed that there was an inverse correlation between concentrations of tNA and mIns at both baseline and at 1 and 3-years follow-up (r = -0.44 to -0.65, p = 0.04 to 0.004). Metabolite concentrations were stable during 1-year follow-up independently of disease activity. Higher concentrations of the astrogliosis marker mIns in MS compared to controls, the inverse relation between MS disease duration and the neuroaxonal integrity marker tNA, as well as the consistent inverse relation between these two metabolites during follow-up, showed that non-lesional white matter pathology is present in this cohort of MS patients in early disease stages. However, metabolite concentrations during follow-up were generally stable and did not reflect differences in disease activity among patients.

Cobaltabis(Dicarbollide) [o-COSAN]- for Boron Neutron Capture Therapy of Head and Neck Cancer: Biodistribution and Irradiation Studies in an Experimental Oral Cancer Model.

Boron neutron capture therapy (BNCT) is a tumor-selective particle radiotherapy that combines preferential boron accumulation in tumors and neutron irradiation. Based on previous studies in tumor-bearing mice, this study evaluated the biodistribution of the sodium salt of cobaltabis(dicarbollide) (Na[3,3'-Co(C2B9H11)2], abbreviated as Na[o-COSAN]) in the hamster cheek pouch oral cancer model and the Na[o-COSAN]/BNCT therapeutic effect on tumors and induced radiotoxicity. The synthesis and comprehensive characterization of 10B-enriched trimethylammonium salt of nido-[7,8-C210B9H12]-o-carborane, along with the cesium and sodium salts of [o-10COSAN] cobaltabis(dicarbollide) are reported here for the first time. Hamsters bearing tumors were injected with Na[o-COSAN] (7.5 mg B/kg) and euthanized at different time-points after injection (30 min, 2, 3, 5, and 18 h post-administration) to evaluate boron uptake in different tissues/organs. Based on these results, tumor-bearing animals were treated with Na[10B-o-COSAN]/BNCT (7.5 mg B/kg b.w., 3 h), prescribing 5 Gy total in absorbed dose to the precancerous tissue surrounding tumors, i.e., the dose-limiting tissue. Na[o-10COSAN] exhibited no toxicity. Although biodistribution studies employing Na[o-COSAN] have shown low absolute boron concentration in the tumor (approx. 11 ppm), Na[o-10COSAN]/BNCT induced a high and significant therapeutic effect on tumors versus the control group (cancerized, untreated animals). Moreover, only half of the animals exhibited severe mucositis in the precancerous dose-limiting tissue after BNCT, which resolved completely at 21 days after irradiation. Na[o-10COSAN] would be potentially useful to treat head and neck cancer with BNCT.

Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs ≤ 4.2%) and concordant (inter-laboratory CVs < 14%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

Ambient Air Pollution and Hospitalizations for Schizophrenia in China

Schizophrenia episodes may be triggered by short-term environmental stimuli. Short-term increases in ambient air pollution levels may elevate the risk of schizophrenia episodes, yet few epidemiologic studies have examined this association. To investigate whether short-term increases in air pollution levels are associated with an additional risk of schizophrenia episodes, independent of absolute air pollution concentrations, and whether sustained increases in air pollution levels for several days are associated with more pronounced risks of schizophrenia episodes. This nationwide, population-based, time-stratified case-crossover study was performed based on hospitalization records for schizophrenia across 295 administrative divisions of prefecture-level or above cities in China. Records were extracted from 2 major health insurance systems from January 1, 2013, to December 31, 2017. Thirty-six cities with a small number of schizophrenia hospitalizations (n < 50) were excluded. Data analysis for this study was performed from January to March 2024. Daily absolute concentrations of fine particulate matter (PM2.5), inhalable particulate matter (PM10), nitrogen dioxide, sulfur dioxide, ozone, and carbon monoxide were collected. Air pollution increases between neighboring days (APINs) were generated as the differences in absolute air pollution concentrations on the current day minus that on the previous day. Sustained increases (APIN ≥5 μg/m3 for PM2.5 and PM10, APIN ≥1 μg/m3 for nitrogen dioxide and sulfur dioxide, and APIN ≥0.05 mg/m3 for carbon monoxide) lasting for 1 or more to 4 or more days were defined for different air pollutants. Patients with schizophrenia episodes were identified by principal discharge diagnoses of schizophrenia. A conditional logistic regression model was used to capture the associations of absolute concentrations, APINs, and sustained increase events for different air pollutants with risks of schizophrenia hospitalizations. The study included 817 296 hospitalization records for schizophrenia across 259 Chinese cities (30.6% aged 0-39 years, 56.4% aged 40-64 years, and 13.0% aged ≥65 years; 55.04% male). After adjusting for the absolute concentrations of respective air pollutants, per-IQR increases in 6-day moving average (lag0-5) APINs of PM2.5, PM10, nitrogen dioxide, sulfur dioxide, and carbon monoxide were associated with increases of 2.37% (95% CI, 0.88%-3.88%), 2.95% (95% CI, 1.46%-4.47%), 4.61% (95% CI, 2.93%-6.32%), 2.16% (95% CI, 0.59%-3.76%), and 2.02% (95% CI, 0.39%-3.68%) in schizophrenia hospitalizations, respectively. Greater risks of schizophrenia hospitalizations were associated with sustained increases in air pollutants lasting for longer durations up to 4 or more days. This case-crossover study of the association between ambient air pollution increases and schizophrenia hospitalizations provides novel evidence that short-term increases in ambient air pollution levels were positively associated with an elevated risk of schizophrenia episodes. Future schizophrenia prevention practices should pay additional attention to APINs, especially sustained increases in air pollution levels for longer durations, besides the absolute air pollution concentrations.

Pharmacokinetic interaction of quetiapine and lamotrigine – victim and perpetrator?

ABSTRACT Objective We aimed to investigate the ambiguous findings of earlier research regarding the reduction of quetiapine plasma levels when combined with lamotrigine, most likely via UDP-glucuronosyltransferase induction by lamotrigine. Methods One thousand one hundred and fifty samples, divided into four groups of patients receiving either quetiapine immediate- (IR) or extended-release (XR) without or in combination with lamotrigine were compared regarding absolute and dose-adjusted plasma concentrations. Furthermore, samples of intra-individual controls were analyzed. Results Patients receiving quetiapine IR in combination with lamotrigine showed 31% lower plasma (p = 0.002) and 23% lower dose-adjusted plasma concentrations (p = 0.004) compared to those receiving IR monotherapy. The proportion of patients with quetiapine plasma concentrations below the lower limit of the therapeutic reference range was 50% and 30% in the combination group and in patients receiving monotherapy, respectively (p = 0.03). However, no significant differences regarding plasma concentration (p = 0.13) and dose-adjusted plasma concentration (p = 0.42) were observed in patients with combination vs. monotherapy with the XR formulation of quetiapine. In the intra-individual controls, no trends could be identified, possibly due to insufficient number of samples (p > 0.05). Conclusions The combination of quetiapine IR with lamotrigine is associated with significantly lower drug concentrations of quetiapine, potentially impacting quetiapine effectiveness. For quetiapine ER, a significant interaction is less likely.

Enantioselective-GCxGC determination of α-terpinyl ethyl ether in wine: Quantitative analysis and identification of main terpene precursors

The present study investigates the presence of α-terpinyl ethyl ether in wine and gives insights on the chemical processes leading to its formation. The analytical determination of (S)-α-terpinyl ethyl ether and (R)-α-terpinyl ethyl ether enantiomers was obtained by enantioselective comprehensive two-dimensional gas chromatography. Applying the two isomers as variables in combination with closely related terpenes, an accurate classification model of wines for the grape variety was successfully applied to a representative set of single-variety wine samples. Although presenting relatively low absolute concentrations, α-terpinyl ethyl ether (along with α-terpineol) resulted to be an inevitable and irreversible degradation product of linalool. In fact, a conversion study from enantiomerically pure (R)-linalool showed a major loss of the initial chiral configuration, i.e. only a very small enantiomeric excess characterized the product. α-Terpineol itself was also confirmed to be a precursor of α-terpinyl ethyl ether, however this process showed a smaller conversion over two weeks than from linalool, and without losses of the initial chiral configuration. In the real samples, the concentration of α-terpinyl ethyl ether was found to be much lower than that of α-terpineol, regardless of the alcohol-to-water ratio. Finally, olfactory descriptors were qualitatively attributed to each α-terpinyl ethyl ether enantiomer.

Fentanyl, Heroin, Methamphetamine, and Cocaine Analyte Concentrations in Urine Drug Testing Specimens

The US is experiencing a protracted drug overdose crisis primarily associated with exposure to illicitly manufactured fentanyl (IMF), methamphetamine, and cocaine. Overdose risk and treatment responses may be directly affected by absolute drug exposure concentrations and drug use prevalence. To quantify changes in absolute drug exposure concentrations from 2013 to 2023. This cross-sectional study analyzed urine drug testing (UDT) results from urine specimens collected between January 1, 2013, and August 22, 2023, in 49 states and the District of Columbia. Urine specimens were obtained from patients aged 18 years or older who presented to substance use disorder treatment clinics. The UDT was ordered by clinicians based on medical necessity. Urine specimens were analyzed for the following drugs or metabolites (analytes tested in parentheses): fentanyl (fentanyl), heroin (6-monoacetylmorphine), cocaine (benzoylecgonine), and methamphetamine (methamphetamine) using liquid chromatography with tandem mass spectrometry. Relative concentrations of fentanyl, heroin, cocaine, and methamphetamine. Creatinine-normalized drug concentration values were log-transformed prior to visualization and statistical analyses. The Mann-Kendall trend test was performed to examine trends over time. To estimate the geospatial and temporal patterns of drug concentration, a second series of models (1 for each drug) with an interaction effect for clinic location and collection year were fit. A total of 921 931 unique UDT samples were collected from patients (549 042 males [59.6%]; median [IQR] age, 34 [27-44] years). The adjusted fentanyl concentration in urine specimens was 38.23 (95% CI, 35.93-40.67) ng/mg creatinine in 2023 and 4.61 (95% CI, 3.59-5.91) ng/mg creatinine in 2013. The adjusted methamphetamine concentration was 3461.59 (95% CI, 3271.88-3662.30) ng/mg creatinine in 2023 and 665.27 (95% CI, 608.51-727.32) ng/mg creatinine in 2013. The adjusted cocaine concentration was 1122.23 (95% CI, 1032.41-1219.87) ng/mg creatinine in 2023 and 559.71 (95% CI, 524.69-597.06) ng/mg creatinine in 2013. The adjusted heroin concentration was 58.36 (95% CI, 48.26-70.58) ng/mg creatinine in 2023 and 146.59 (95% CI, 136.06-157.92) ng/mg creatinine in 2013. Drug concentrations varied across US Census divisions. This cross-sectional study found that absolute concentrations of fentanyl, methamphetamine, and cocaine in urine specimens increased from 2013 to 2023, with a decrease in heroin concentration during that period. The findings suggest that exposure to these substances, as well as the illicit drug supply, has fundamentally changed in many parts of the US, highlighting the need to reinforce surveillance initiatives and accelerate efforts to treat individuals with IMF and/or stimulant exposure.

Quantifying the extended energy metabolome of industrially important microorganisms (Saccharomyces cerevisiae) using ultra-performance liquid chromatography with mass spectrometry

This study has developed a new targeted methodology for the separation, detection, and quantification of metabolites from the wider energy metabolome of industrially important microorganisms such as Saccharomyces cerevisiae in a single analytical sample. This has been achieved using UHPLC-MS technology in HILIC mode. Absolute concentrations of metabolites nicotinamide adenine dinucleotide (NAD), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate (NADP), nicotinamide adenine dinucleotide phosphate reduced (NADPH), flavin adenine dinucleotide (FAD), adenosine-monophosphate (AMP), adenosine-diphosphate (ADP), and adenosine-triphosphate (ATP) were determined in a single extraction and analytical methodology.This study demonstrated the development of a rapid, statistically robust, and reproducible methodology through regression calibrations of standard samples from 0.1 to 100 µMol providing a correlation value of r2 = >0.98 for all metabolites. Sample preparation, extraction and analytical methodologies used showed high accuracy, sensitivity, and recovery. With an LOD and LOQ for the targeted analysis of metabolites from the wider energy metabolism in a single sample and analytical run with the lowest LOD of 0.055 nMol (±0.002) and lowest LOQ of 0.167 nMol (±0.006). This method was then applied to Saccharomyces cerevisiae cell culture to evaluate the methodology in industrially used microbial cultures. Results obtained have been statistically determined to be robust and reproducible through recovery analysis using deuterated and isotopically labelled internal standards AMP-15N, ADP-15N and ATP-d14.

Dynamics of antibiotic resistance agents during sludge alkalinization treatment

This study aimed to assess the removal of antimicrobial resistance agents (antibiotics, antibiotic-resistant bacteria - ARB, and antimicrobial resistance genes - ARGs) from aerobic and anaerobic sludges treated with quicklime (chemical alkalinization). Different mixing ratios (25%, 35%, and 45%) and contact times (2 h and 72 h) were evaluated. The findings revealed that anaerobic sludge responded more effectively to alkaline treatment, achieving better removal rates of antibiotics, ARB, and ARGs compared to aerobic sludge. The 45% lime treatment yielded the highest antibiotic removal rates, with average reductions of 19% in aerobic sludge and 28% in anaerobic sludge. The 35% lime treatment was the most effective in reducing ARGs across both types of sludge (average removal of 2 logs). The 25% lime treatment proved most efficient for removing ARB, with average reductions of 4 logs (aerobic) and 5 logs (anaerobic). The contact time between the sludge and quicklime also influenced the removal of resistance agents. An increase in the proportion of antibiotics and the absolute concentration of ARB and ARGs was observed after 72 h compared to the samples analyzed after 2 h of contact. This increase was more pronounced in aerobic sludge samples treated with 35% and 45% lime. Despite the overall reduction, none of the monitored resistant genes or bacteria were completely eradicated in both sludge samples, raising concerns about their potential dissemination into the environment.

Absolute concentration robustness: Algebra and geometry

Absolute concentration robustness: Algebra and geometry

Analysis of Total Microcystins using LC-MS/MS: Method Improvements and Sample Applications

Microcystin variants (MCs), including microcystin-LR (MC-LR) and related variants, are produced by cyanobacteria and are regulated due to their potential health risks. Current methods for MC analysis predominantly employ LC-MS/MS, limited by the availability of reference materials necessary for quantification. As an alternative method for total MC quantification, the analysis of 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), formed by oxidation of the common ADDA structure in MCs, offers economic and practical advantages by avoiding the need for multiple MC reference materials. This method allows total MC quantification across various matrices, independent of specific MC variants. In this study, we evaluated the utility of MMPB-d3 as a surrogate to assess recovery rates throughout the processes critical for MMPB quantification. Comparative analysis with 4-phenylbutyric acid (4-PB) highlighted the stability of MMPB-d3 under oxidative conditions, which is critical for its application as a surrogate or internal standard. The oxidation process was optimized to achieve consistent recovery rates, averaging 101%, ensuring reliable conversion of MCs to MMPB for subsequent analysis. The limit of quantification (LOQ) has been improved due to the large volume injection of up to 0.1 µg/L. Comparison of MMPB concentrations from field samples with conventional LC-MS/MS results showed a significant correlation (r = 0.80, p &lt; 0.005), but with variations in the absolute concentrations. Calculations using a formulation model with MMPB concentrations allowed us to estimate total MC levels, demonstrating the potential of the proposed method to complement existing target measurement techniques.

Integrating HRMAS-NMR Data and Machine Learning-Assisted Profiling of Metabolite Fluxes to Classify Low- and High-Grade Gliomas.

Diagnosing and classifying central nervous system tumors such as gliomas or glioblastomas pose a significant challenge due to their aggressive and infiltrative nature. However, recent advancements in metabolomics and magnetic resonance spectroscopy (MRS) offer promising avenues for differentiating tumor grades both in vivo and ex vivo. This study aimed to explore tissue-based metabolic signatures to classify/distinguish between low- and high-grade gliomas. Forty-six histologically confirmed, intact solid tumor samples from glioma patients were analyzed using high-resolution magic angle spinning nuclear magnetic resonance (HRMAS-NMR) spectroscopy. By integrating machine learning (ML) algorithms, spectral regions with the most discriminative potential were identified. Validation was performed through univariate and multivariate statistical analyses, along with HRMAS-NMR analyses of 46 paired plasma samples. Amongst the various ML models applied, the logistics regression identified 46 spectral regions capable of sub-classifying gliomas with accuracy 87% (F1-measure 0.87, Precision 0.82, Recall 0.93), whereas the extra-tree classifier identified three spectral regions with predictive accuracy of 91% (F1-measure 0.91, Precision 0.85, Recall 0.97). Wilcoxon test presented 51 spectral regions significantly differentiating low- and high-grade glioma groups (p < 0.05). Based on sensitivity and area under the curve values, 40 spectral regions corresponding to 18 metabolites were considered as potential biomarkers for tissue-based glioma classification and amongst these N-acetyl aspartate, glutamate, and glutamine emerged as the most important markers. These markers were validated in paired plasma samples, and their absolute concentrations were computed. Our results demonstrate that the metabolic markers identified through the HRMAS-NMR-ML analysis framework, and their associated metabolic networks, hold promise for targeted treatment planning and clinical interventions in the future.

Challenges and advances for huntingtin detection in cerebrospinal fluid: in support of relative quantification.

Huntington disease (HD) is a progressive and devastating neurodegenerative disease caused by expansion of a glutamine-coding CAG tract in the huntingtin (HTT) gene above a critical threshold of ~35 repeats resulting in expression of mutant HTT (mHTT). A promising treatment approach being tested in clinical trials is HTT lowering, which aims to reduce levels of the mHTT protein. Target engagement of these therapies in the brain are inferred using antibody-based assays to measure mHTT levels in the cerebrospinal fluid (CSF), which is frequently reported as absolute mHTT concentration based on a monomeric protein standard used to generate a standard curve. However, patient biofluids are a complex milieu of different mHTT protein species, suggesting that absolute quantitation is challenging, and a single, recombinant protein standard may not be sufficient to interpret assay signal as molar mHTT concentration. In this study, we used immunoprecipitation and flow cytometry (IP-FCM) to investigate different factors that influence mHTT detection assay signal. Our results show that HTT protein fragmentation, protein-protein interactions, affinity tag positioning, oligomerization and polyglutamine tract length affect assay signal intensity, indicating that absolute HTT quantitation in heterogeneous biological samples is not possible with current technologies using a single standard protein. We also explore the binding specificity of the MW1 anti-polyglutamine antibody, commonly used in these assays as a mHTT-selective reagent and demonstrate that mHTT binding is preferred but not specific. Furthermore, we find that MW1 depletion is not only incomplete, leaving residual mHTT, but also non-specific, resulting in pull down of some wildtype HTT protein. Based on these observations, we recommend that mHTT detection assays report only relative mHTT quantitation using normalized arbitrary units of assay signal intensity, rather than molar concentrations, in the assessment of central nervous system HTT lowering in ongoing clinical and preclinical studies, and that MW1-depletion not be used a method for quantifying wildtype HTT protein.

Analysis Pork Contamination in Beef Meatballs through Polymerase Chain Reaction in Palu City

The need for halal products is essential, particularly among various processed beef products like meatballs, which are popular among nearly the entire community. These products are susceptible to issues of meat counterfeiting for greater economic gain. This study aims to determine the smallest DNA concentration that can still be detected and to assess whether there is any pork contamination in meatballs circulating in Palu City using Polymerase Chain Reaction (PCR). This test uses four DNA samples obtained from fresh meat to test the specificity of the primer: wild boar, rat, and goat DNA samples as comparisons, and pig DNA as a positive control. The presence of DNA bands in the agarose gel electrophoresis visualization results indicates that DNA amplification has occurred, meaning that the sample can be detected. The analysis results showed that the primer used (Cyt-b) had good specificity for detecting pork DNA, while the absolute pork DNA concentration of 0.01 ng/µL and the reference meatball DNA with a concentration of 0.01% are the smallest limits that can still be detected using PCR. Of the four meatball samples from Palu City, one sample produced a DNA band, indicating that the sample contains or is contaminated with pig DNA. This method is valid as it meets the specificity test and demonstrates a good detection limit.

Anthropic-Induced Variability of Greenhouse Gasses and Aerosols at the WMO/GAW Coastal Site of Lamezia Terme (Calabria, Southern Italy): Towards a New Method to Assess the Weekly Distribution of Gathered Data

The key to a sustainable future is the reduction in humankind’s impact on natural systems via the development of new technologies and the improvement in source apportionment. Although days, years and seasons are arbitrarily set, their mechanisms are based on natural cycles driven by Earth’s orbital periods. This is not the case for weeks, which are a pure anthropic category and are known from the literature to influence emission cycles and atmospheric chemistry. For the first time since it started data gathering operations, CO (carbon monoxide), CO2 (carbon dioxide), CH4 (methane) and eBC (equivalent black carbon) values detected by the Lamezia Terme WMO/GAW station in Calabria, Southern Italy, have been evaluated via a two-pronged approach accounting for weekly variations in absolute concentrations, as well as the number of hourly averages exceeding select thresholds. The analyses were performed on seven continuous years of measurements from 2016 to 2022. The results demonstrate that the analyzed GHGs (greenhouse gasses) and aerosols respond differently to weekly cycles throughout the seasons, and these findings provide completely new insights into source apportionment characterization. Moreover, the results have been combined into a new parameter: the hereby defined WDWO (Weighed Distribution of Weekly Outbreaks) normalizes weekly trends in CO, CO2, CH4 and eBC on an absolute scale, with the scope of providing regulators and researchers alike with a new tool meant to better evaluate anthropogenic pollution and mitigate its effects on the environment and human health.

Butterfly species vary in sex‐specific sodium accumulation from larval diets

Abstract Sodium is essential for animals, and its heterogeneous distribution can cause a range of phenomena, from sodium‐seeking behaviours to impacting their performance. Although sodium content in soils and plants is relatively well documented, data for higher trophic levels are limited. Knowledge of the variation in sodium in lower trophic levels could have implications for understanding the behaviour and physiology of species at higher levels. We investigated the variation in tissue sodium concentration between males and females of four butterfly species. Puddling behaviour of Lepidoptera suggests sodium needs of males are generally greater than females, thus, we predicted males would accumulate more sodium than females on a given diet. Larvae were reared on plants (for Battus philenor, Chlosyne lacinia and Danaus plexippus) and an artificial diet (for Pieris rapae) under Low Na (no added sodium) and High Na (sodium added) conditions. Among species and sexes, we quantified and compared adult absolute tissue sodium concentrations and bioconcentration factors, which indicate net sodium accumulation or excretion relative to individuals' diets. On average, individuals on low‐sodium diets had higher bioconcentration values across all species. Male butterflies accumulated significantly higher sodium concentrations than females in two sodium treatments for B. philenor, and P. rapae and only in the High Na treatment for C. lacinia. However, in D. plexippus, individuals accumulate sodium in the High Na treatment, but males and females responded in the same way. Our study revealed sex‐ and species‐specific patterns of butterfly sodium accumulation, which could be linked to variations in behaviour and/or performance. Differences in sodium content across species have implications for variation in predation and trophic‐level interactions, an interesting avenue for future ecological and evolutionary research.

Standardization of Dual-Energy CT Iodine Uptake of the Abdomen and Pelvis: Defining Reference Values in a Big Data Cohort.

Background: To establish dual-energy-derived iodine density reference values in abdominopelvic organs in a large cohort of healthy subjects. Methods: 597 patients who underwent portal venous phase dual-energy CT scans of the abdomen were retrospectively enrolled. Iodine distribution maps were reconstructed, and regions of interest measurements were placed in abdominal and pelvic structures to obtain absolute iodine values. Subsequently, normalization of the abdominal aorta was conducted to obtain normalized iodine ratios. The values obtained were subsequently analyzed and differences were investigated in subgroups defined by sex, age and BMI. Results: Overall mean iodine uptake values and normalized iodine ratios ranged between 0.31 and 6.08 mg/mL and 0.06 and 1.20, respectively. Women exhibited higher absolute iodine concentration across all organs. With increasing age, normalized iodine ratios mostly tend to decrease, being most significant in the uterus, prostate, and kidneys (p < 0.015). BMI was the parameter less responsible for variations in iodine concentrations; normal weighted patients demonstrated higher values of both absolute and normalized iodine. Conclusions: Iodine concentration values and normalized iodine ratios of abdominal and pelvic organs reveal significant gender-, age-, and BMI-related differences, underscoring the necessity to integrate these variables into clinical practice.

99 The Effects of β-caryophyllene on butyrate utilization and metabolism in Caco 2 cells

Abstract Butyrate is important for regulating energy status and cellular metabolism. Beta-caryophyllene (BCP) is a plant compound which may exert potentially beneficial effects on intestinal epithelial cells, such as barrier integrity and modulation of nutrient utilization. The goal of this preliminary study was to investigate the effects of BCP on butyrate utilization and metabolism in intestinal epithelial cells. Caco 2 cells were used as a model for the intestinal barrier. Cells were seeded in multi-well plates with hanging inserts (n = 4) and grown for 18 d. On d 19, 1 of 4 treatments was added to triplicate wells per plate. Treatments included vehicle control (VC), 40 µM BCP, 2 mM butyrate (BTR), and butyrate plus BCP (BB). Treatments were added to the apical side and incubated for 24 and 48 h. Transepithelial electrical resistance (TEER; ohms/cm2) readings and supernatant samples from both the apical and basolateral sides of the cell layer were taken at each time point, and butyrate and β- hydroxybutyrate (BHB) concentrations measured. Butyrate utilization was calculated as absolute nmol lost. Statistical analyses were performed using lm and emmeans packages of R, with treatment, time and side (of cell layer) as fixed effects. All wells started with the same statistical TEER values on h 0. After 24 and 48 h BCP showed no changes in TEER (P &amp;gt; 0.05). Compared with VC, the mean TEER for BTR and BB was greater at 24 h (579 vs 878 and 1,008 ± 35, respectively; P &amp;lt; 0.001). After 48 h, all groups returned to baseline, except BB which had a greater TEER (883 ± 35; P &amp;lt; 0.001) compared with other treatments. After 24 and 48 h BCP showed no changes for both sides of the cell layer (P &amp;gt; 0.05). Overall, absolute BHB concentrations increased from 24 to 48 h in all treatments (P &amp;lt; 0.05). Production of BHB in each of the VC and BC groups was less than 15 nmol at each time point. For BB and BTR groups, more nmol BHB was detected on the basolateral side (31.9 and 22.2 ± 1.3) vs apical (14.0 and 10.4 ± 1.3, respectively; P &amp;lt; 0.001) after 24 h. After 48 h, BHB was also greater on the basolateral side (58.2 and 45.2 ± 1.3 nmol for BB and BUT, respectively) vs apical side (30.2 and 24.0 ± 1.3 nmol; P &amp;lt; 0.001). When looking at BTR utilization, the BB treatment had greater butyrate disappearance than BTR in 24 h (254 vs 169 ± 12.6 nmol; P = 0.0005) and 48h (707 vs 573 ± 12.6 nmol; P &amp;lt; 0.0001). BCP may promote the metabolism of butyrate by intestinal epithelial cells. More research is needed to understand the mechanism of how BCP increases cellular butyrate utilization.

Longitudinal Analysis of Tear Cytokine Ratios in Association with Dry Eye Signs and Symptoms in the Dry Eye Assessment and Management (DREAM) Study

Purpose To extend cross-sectional data on cytokine ratios (CRs) in dry eye disease (DED) signs by investigating longitudinal change in pro- to anti-inflammatory CRs and associations with change in DED signs and symptoms. Methods Secondary analysis of fifty-four subjects [mean age 57.3 (SD 13.2) years, 85.2% female; 68.5% white] with ≥ 4 uL pooled tear volumes at months 0, 6, and 12. Pro-inflammatory (IL-1b, IL-6, IL-8, IL-17A, IFN-g, and TNF-a) to anti-inflammatory (IL-6, IL-10) cytokine ratios (CR) were calculated. DED signs (corneal and conjunctival staining scores, tear break-up time, Schirmer test, Meibomian gland plugging, tear osmolarity, composite sign severity score) and symptoms [Ocular Surface Disease Index (OSDI)] were measured. Changes over time in DED signs, symptoms, and CRs were evaluated using longitudinal models. Correlations between changes in CR and changes in DED signs and symptoms were evaluated using Spearman correlation coefficients (rho). Results DED signs which improved over time (p < 0.001) included corneal and conjunctival staining score, tear break-up time, tear osmolarity, and composite sign severity score. Using IL-10 as anti-inflammatory, changes in corneal and conjunctival staining and composite severity score significantly correlated with changes in pro- to anti-inflammatory CRs from month 0 to 6 (|rho|: 0.29-0.45, p: 0.003-0.04) but not between month 0 to 12 (|rho|: 0.01 to 0.24, all p > 0.08). DED symptoms decreased across one year (p < = 0.001) for all OSDI measures; these changes did not correlate with changes in CRs (|rho|: 0.00 to 0.29, all p > 0.04). Conclusions Improvement in some DED signs across one year correlated weakly with decreases in pro- to anti-inflammatory CRs, in alignment with the understanding of DED as inflammatory. CRs may provide greater insight than absolute tear cytokine concentrations as possible DED biomarkers. Additional studies that provide standardized clinical information and tear collection and analysis are needed to validate CRs in DED.