Temporal dynamics of drinking water sodium levels in coastal areas, Cyprus 2009-2020.

Around the world, groundwater salinity levels are increasing in coastal areas, as a result of its systematic overexploitation for domestic, agricultural and industrial demand and potentially due to climate change manifestations (such as, sea level rise). We hypothesized that the groundwater quality of many Mediterranean coastal areas is already being perturbed, especially for water salinity, depending on the groundwater distance from the seafront. The objectives of this study were: i) to evaluate the magnitude and temporal variance of drinking water sodium (Na) as a metric of salt intake used for public health purposes using drinking water data in Cyprus; and ii) to examine the degree of Na enrichment in drinking water as defined by the seawater coastline distance of each sampling point. Open access governmental data of drinking water Na (n = 3304), daily max ambient air temperature and total rainfall were obtained for the period of 2009-2020 from governmental repositories. Linear mixed-effect regression models of drinking water Na with unsupervised covariance matrix were used. After adjusting for temperature and rainfall data, there was a significant annual increase in drinking water Na levels over time (beta = 0.01; 95 % CI: 0.00, 0.02; p = 0.02) for the coastal areas (<10 km from coastline, cutoff used by the EU Environment Agency), but this was not the case for non-coastal areas (>10 km distance from coastline). The distance of each sampling point from the coastline in Cyprus was negatively associated with drinking water Na in coastal areas (beta = -0.04, 95%CI: -0.06, -0.01; p = 0.002); this was not the case for non-coastal areas. More research is warranted to better understand the impacts of global environmental change on water quality in association with the burden of disease in coastal areas.

Vitamin D: Analytical Advances, Clinical Impact, and Ongoing Debates on Health Perspectives.

BACKGROUND: Vitamin D, acknowledged since the 1930s for its role in preventing rickets, gained additional prominence in relation to fragility fracture prevention in the late 1980s. From the early 2000s, connections between vitamin D deficiency and extra-skeletal pathologies emerged, alongside increased awareness of widespread deficits. This prompted crucial debates on optimal serum concentrations, expected to conclude when the outcomes of high-dose supplementation randomized controlled trials were available. Skepticism arose with inconclusive results from these trials. CONTENT: This review begins with an exploration of vitamin D metabolism, followed by a detailed description of the measurement of vitamin D metabolites and the crucial role of standardization. Subsequent sections focus on the association of vitamin D with bone health and explore the extra-skeletal effects. The review concludes with a comprehensive discussion on the definition of vitamin D status and its implications for supplementation. SUMMARY: Despite standardization efforts, assay variations and challenges still exist, especially in specific patient groups. Vitamin D supplementation has a significant impact on bone metabolism and optimal vitamin D status improves the efficacy of antiresorptive drugs such as bisphosphonates. The extra-skeletal effects of vitamin D remain debated, but may include potential benefits in conditions such as respiratory infections and cancer mortality, particularly in deficient individuals. The definition of vitamin D sufficiency is nuanced, especially when variations in population groups and analytical methods are taken into account. Despite ongoing debates and recent mega-trials tempering enthusiasm, vitamin D remains a complex and essential element in human health. Further research is needed to clarify its role in various health outcomes and guide supplementation strategies.

An anti-inflammatory response of an organic food intervention by reducing pesticide exposures in children of Cyprus: A cluster-randomized crossover trial.

Organic food consumption in children has been shown to reduce the body burden of chemical pesticides. However, there is little evidence of human health benefits associated with the consumption of organic foods. The objectives were to i) determine the effectiveness of an organic food intervention treatment in reducing the magnitude of an inflammation biomarker (C-reactive protein, CRP) in children (10-12 years) and ii) assess the association between the urinary biomarkers of exposure to pesticides and CRP. This work was part of the ORGANIKO cluster-randomized cross-over trial entailing a 40-day organic food treatment in healthy children. Urinary biomarkers of exposure to pesticides and inflammation (CRP) were measured using tandem mass spectrometry and ELISA immunoassay, respectively. Linear mixed-effect regression models of CRP were used to account for the effect and duration of organic food treatment. Multiple comparisons were handled using Benjamini-Hochberg correction. Results supported an anti-inflammatory effect of organic food treatment in children, albeit with mixed results, depending on the creatinine adjustment method; biomarker levels were divided by urinary creatinine (method a1), or urinary creatinine was used as a fixed effect variable (a2). In the a1 method, a time-dependent reduction for creatinine-adjusted CRP (ß = -0.019; 95% CI: -0.031, -0.006; q = 0.045) was observed during the organic food intervention period. A statistically significant association (ß = 0.104; 95% CI: 0.035, 0.173; q = 0.045) was found between the biomarker of pyrethroids exposure (3-PBA) and CRP inflammatory biomarker, but not for 6-CN. In the a2 method, similar trend of time-dependent reduction for creatinine-adjusted CRP (ß = -0.008; 95% CI: -0.021, 0.004; p = 0.197) was observed during the organic food intervention period, but did not reach statistical significance (q > 0.05); the associations of pyrethroid and neonicotinoid biomarkers with CRP were not statistically significant (q > 0.05). More studies are warranted to sufficiently understand the potential anti-inflammatory response of an organic food treatment.

Decoding the exposome: data science methodologies and implications in exposome-wide association studies (ExWASs).

This paper explores the exposome concept and its role in elucidating the interplay between environmental exposures and human health. We introduce two key concepts critical for exposomics research. Firstly, we discuss the joint impact of genetics and environment on phenotypes, emphasizing the variance attributable to shared and nonshared environmental factors, underscoring the complexity of quantifying the exposome's influence on health outcomes. Secondly, we introduce the importance of advanced data-driven methods in large cohort studies for exposomic measurements. Here, we introduce the exposome-wide association study (ExWAS), an approach designed for systematic discovery of relationships between phenotypes and various exposures, identifying significant associations while controlling for multiple comparisons. We advocate for the standardized use of the term "exposome-wide association study, ExWAS," to facilitate clear communication and literature retrieval in this field. The paper aims to guide future health researchers in understanding and evaluating exposomic studies. Our discussion extends to emerging topics, such as FAIR Data Principles, biobanked healthcare datasets, and the functional exposome, outlining the future directions in exposomic research. This abstract provides a succinct overview of our comprehensive approach to understanding the complex dynamics of the exposome and its significant implications for human health.

Comparator Data Characteristics and Testing Procedures for the Clinical Performance Evaluation of Continuous Glucose Monitoring Systems.

Comparing the performance of different continuous glucose monitoring (CGM) systems is challenging due to the lack of comprehensive guidelines for clinical study design. In particular, the absence of concise requirements for the distribution of comparator (reference) blood glucose (BG) concentrations and their rate of change (RoC) that are used to evaluate CGM performance, impairs comparability. For this article, several experts in the field of CGM performance testing have collaborated to propose characteristics of the distribution of comparator measurements that should be collected during CGM performance testing. Specifically, it is proposed that at least 7.5% of comparator BG concentrations are <70 mg/dL (3.9 mmol/L) and >300 mg/dL (16.7 mmol/L), respectively, and that at least 7.5% of BG-RoC combinations indicate fast BG changes with impending hypo- or hyperglycemia, respectively. These proposed characteristics of the comparator data can facilitate the harmonization of testing conditions across different studies and CGM systems and ensure that the most relevant scenarios representing real-life situations are established during performance testing. In addition, a study protocol and testing procedure for the manipulation of glucose levels are suggested that enable the collection of comparator data with these characteristics. This work is an important step toward establishing a future standard for the performance evaluation of CGM systems.

Assessing the status of European laboratories in evaluating biomarkers for chronic kidney diseases (CKD) and recommendations for improvement: insights from the 2022 EFLM Task Group on CKD survey.

OBJECTIVES: Chronic kidney disease (CKD) is a global health issue, ranking as the third leading cause of death worldwide. CKD diagnosis and management depend on clinical laboratory tests, necessitating consistency for precise patient care. Global harmonization of CKD testing through clinical practice guidelines (CPGs) is recommended. Prior to CPG development, assessing the current CKD testing landscape is crucial. In 2022, the European Federation of Laboratory Medicine (EFLM) conducted an online survey among European laboratories associated with EFLM, evaluating CKD testing practices, including new glomerular filtration rate (GFR) estimation methods. This report summarizes the 2022 survey findings and offers recommendations for improving CKD test standardization. METHODS: An online survey was conducted in November 2022 using a questionnaire hosted on LimeSurvey sent to European laboratories affiliated with the EFLM. The survey results were recorded in Excel files and analysed. RESULTS: The results highlight significant discrepancies among countries in unit expression, methods, cystatin C use, and GFR calculation equations. Additionally, limited attention to pediatric renal biology specifics, varied proteinuria and albuminuria result expressions, and limited awareness of GFR measurement methods through iohexol clearance are noted. CONCLUSIONS: In an effort to enhance the standardization of crucial biomarkers utilized in nephrology for evaluating renal function and diagnosing kidney injuries, the EFLM Task Group on CKD suggests nine practical recommendations tailored for European laboratories. The group is confident that implementing these measures will minimize result expression discrepancies, ultimately leading to enhanced patient care.

Objectively measured cognitive dysfunction in patients with primary hyperparathyroidism improves after parathyroidectomy.

BACKGROUND: A relationship between primary hyperparathyroidism (PHPT) and decreased quality of life has been shown using patient-reported outcome measures, including Pasieka's Parathyroid Assessment of Symptoms, SF-36, and PROMIS. Despite this, there remains a paucity of objectively measured data demonstrating cognitive dysfunction in patients with PHPT. We assessed whether parathyroidectomy resulted in quantifiable cognitive improvement. METHODS: We examined 59 consecutive patients with PHPT who underwent parathyroidectomy at a single institution between 2019 and 2021. We used BrainCheck, a clinically validated objective measure of neurocognition, to assess pre- and postoperative neurocognitive changes and evaluated associations between BrainCheck scores and parathyroidectomy using the Wilcoxon signed-rank test. RESULTS: Of the 59 patients with PHPT who underwent parathyroidectomy and rapid cognitive assessment with BrainCheck, 72.9% were female, 49.2% were White, and 30.5% were African American. A total of 44.1% of patients preoperatively showed neurocognitive dysfunction relative to the general population compared to 22% postoperatively, representing an improvement in 53% of the cohort. Postoperative scores for the entire cohort were significantly higher than preoperative scores (Z =2.85, P = .004). This association remained significant when the cohort was stratified by sex, as both males (Z =2.02, P = .044) and females (Z =2.09, P = .037) had a significant increase in scores. Domain sub-analysis demonstrated a significant association between parathyroidectomy and improved executive function (P < .01). CONCLUSION: Patients with PHPT experience objectively measurable cognitive changes associated with PHPT that can be reversed by parathyroidectomy, with improvements observed as early as 2 weeks after surgery. Further research with a larger cohort is needed to corroborate our findings.

Glucose-dependent insulinotropic polypeptide regulates body weight and food intake via GABAergic neurons in mice.

The development of single-molecule co-agonists for the glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) and glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) is considered a breakthrough in the treatment of obesity and type 2 diabetes. But although GIPR-GLP-1R co-agonism decreases body weight with superior efficacy relative to GLP-1R agonism alone in preclinical1-3 and clinical studies4,5, the role of GIP in regulating energy metabolism remains enigmatic. Increasing evidence suggests that long-acting GIPR agonists act in the brain to decrease body weight through the inhibition of food intake3,6-8; however, the mechanisms and neuronal populations through which GIP affects metabolism remain to be identified. Here, we report that long-acting GIPR agonists and GIPR-GLP-1R co-agonists decrease body weight and food intake via inhibitory GABAergic neurons. We show that acyl-GIP decreases body weight and food intake in male diet-induced obese wild-type mice, but not in mice with deletion of Gipr in Vgat(also known as Slc32a1)-expressing GABAergic neurons (Vgat-Gipr knockout). Whereas the GIPR-GLP-1R co-agonist MAR709 leads, in male diet-induced obese wild-type mice, to greater weight loss and further inhibition of food intake relative to a pharmacokinetically matched acyl-GLP-1 control, this superiority over GLP-1 vanishes in Vgat-Gipr knockout mice. Our data demonstrate that long-acting GIPR agonists crucially depend on GIPR signaling in inhibitory GABAergic neurons to decrease body weight and food intake.

High-calorie diets uncouple hypothalamic oxytocin neurons from a gut-to-brain satiation pathway via κ-opioid signaling.

Oxytocin-expressing paraventricular hypothalamic neurons (PVNOT neurons) integrate afferent signals from the gut, including cholecystokinin (CCK), to adjust whole-body energy homeostasis. However, the molecular underpinnings by which PVNOT neurons orchestrate gut-to-brain feeding control remain unclear. Here, we show that mice undergoing selective ablation of PVNOT neurons fail to reduce food intake in response to CCK and develop hyperphagic obesity on a chow diet. Notably, exposing wild-type mice to a high-fat/high-sugar (HFHS) diet recapitulates this insensitivity toward CCK, which is linked to diet-induced transcriptional and electrophysiological aberrations specifically in PVNOT neurons. Restoring OT pathways in diet-induced obese (DIO) mice via chemogenetics or polypharmacology sufficiently re-establishes CCK's anorexigenic effects. Last, by single-cell profiling, we identify a specialized PVNOT neuronal subpopulation with increased κ-opioid signaling under an HFHS diet, which restrains their CCK-evoked activation. In sum, we document a (patho)mechanism by which PVNOT signaling uncouples a gut-brain satiation pathway under obesogenic conditions.

A comparative study between low- and high-tech methods for the detection and mitigation of illicit connections in stormwater systems.

Illicit connections of wastewater to stormwater systems are the main drawback of separate sewer systems, as they lead to a direct discharge of untreated wastewater to the aquatic environment. Consequently, several inspection methods have been developed for detecting illicit connections. This study simultaneously applied several low- and high-tech methods for the detection of illicit connections in the same catchment (De Heuvel, the Netherlands). The methods included mesh wire screens for capturing coarse contamination, measurements of electroconductivity and temperature, sampling and quantification of Escherichia coli and extended-spectrum ß-lactamase-producing E. coli (ESBL-EC), DNA analysis via quantitative polymerase chain reaction for human-, dog-, and bird-specific fecal indicators, and distributed temperature sensing. Significant illicit connections could be identified using all methods. Nonetheless, hydraulic conditions and, predominantly, the sewage volume determine whether a misconnection can be detected by especially the low-tech methods. Using these results, the identified misconnections were repaired and biological and DNA analyses were repeated. Our results demonstrate that there were no changes in E. coli or ESBL-EC before and after mitigation, suggesting that these common markers of fecal contamination are not specific enough to evaluate the performance of mitigation efforts. However, a marked decrease in human wastewater markers (HF183) was observed.

Prevalence and sociodemographic determinants of selected noncommunicable diseases among the general population of Cyprus: A large population-based cross-sectional study.

Background: Noncommunicable diseases (NCDs) are the leading cause of morbidity and mortality worldwide. The aim of this study was to determine the prevalence of NCDs among the population of Cyprus and to identify the distribution of the socioeconomic and demographic determinants among individuals with the most frequent NCDs. Methods: A cross-sectional study was conducted using stratified sampling. Socioeconomic and demographic characteristics and the presence of NCDs were collected through a standardized questionnaire. The diseases were classified using the 10th revision of the International Classification of Diseases (ICD-10). Results: In total, 1140 individuals participated in the study, among whom 590 (51.7%) had at least one chronic disease. The most prevalent NCDs were hyperlipidemia (17.4%), hypertension (12.9%), and thyroid diseases (8.4%). We identified more males than females with hyperlipidemia aged 25-44 years old and >65 years old (p = 0.024), more males compared to females with hypertension (p = 0.001) and more females compared to males with thyroid diseases (p < 0.001). Individuals with hypertension and hyperlipidemia were more likely to be married, to have completed a higher education, and to have a high annual income. Discussion: In Cyprus, the majority of the general population had at least one NCD. Hyperlipidemia, hypertension, and thyroid disease are relatively common, even at younger ages, highlighting the need for the development of public health programs aimed at addressing and preventing NCDs.

Characterization of the internal working-life exposome using minimally and non-invasive sampling methods - a narrative review.

During recent years, we are moving away from the 'one exposure, one disease'-approach in occupational settings and towards a more comprehensive approach, taking into account the totality of exposures during a life course by using an exposome approach. Taking an exposome approach however is accompanied by many challenges, one of which, for example, relates to the collection of biological samples. Methods used for sample collection in occupational exposome studies should ideally be minimally invasive, while at the same time sensitive, and enable meaningful repeated sampling in a large population and over a longer time period. This might be hampered in specific situations e.g., people working in remote areas, during pandemics or with flexible work hours. In these situations, using self-sampling techniques might offer a solution. Therefore, our aim was to identify existing self-sampling techniques and to evaluate the applicability of these techniques in an occupational exposome context by conducting a literature review. We here present an overview of current self-sampling methodologies used to characterize the internal exposome. In addition, the use of different biological matrices was evaluated and subdivided based on their level of invasiveness and applicability in an occupational exposome context. In conclusion, this review and the overview of self-sampling techniques presented herein can serve as a guide in the design of future (occupational) exposome studies while circumventing sample collection challenges associated with exposome studies.

Optical vortices shape optical tornados.

We demonstrate that by seeding an accelerating ring-Airy beam with a finite number of off-axis optical vortices, it transforms into a tornado wave (ToW) upon propagation. Using numerical simulations, we show that both the spiraling high-intensity lobes and the optical vortices exhibit angular acceleration and follow interwinding braid-like trajectories. Likewise, we study the effect of the number, position, and topological charge of the vortices on the propagation dynamics and reveal the connection between optical vortices and optical tornados.

Clinical Performance Evaluation of Continuous Glucose Monitoring Systems: A Scoping Review and Recommendations for Reporting.

The use of different approaches for design and results presentation of studies for the clinical performance evaluation of continuous glucose monitoring (CGM) systems has long been recognized as a major challenge in comparing their results. However, a comprehensive characterization of the variability in study designs is currently unavailable. This article presents a scoping review of clinical CGM performance evaluations published between 2002 and 2022. Specifically, this review quantifies the prevalence of numerous options associated with various aspects of study design, including subject population, comparator (reference) method selection, testing procedures, and statistical accuracy evaluation. We found that there is a large variability in nearly all of those aspects and, in particular, in the characteristics of the comparator measurements. Furthermore, these characteristics as well as other crucial aspects of study design are often not reported in sufficient detail to allow an informed interpretation of study results. We therefore provide recommendations for reporting the general study design, CGM system use, comparator measurement approach, testing procedures, and data analysis/statistical performance evaluation. Additionally, this review aims to serve as a foundation for the development of a standardized CGM performance evaluation procedure, thereby supporting the goals and objectives of the Working Group on CGM established by the Scientific Division of the International Federation of Clinical Chemistry and Laboratory Medicine.

Chrono-modulated effects of external stressors on oxidative stress and damage in humans: A scoping review on night shift work.

BACKGROUND: Oxidative stress and tissue damage (OSD) play a pivotal role as an early-stage process in chronic disease pathogenesis. However, there has been little research to better understand the temporal (χρόνος[chronos]) dimensions of OSD process associated with environmental (non-genetic, including behaviors/lifestyle) and/or occupational stressors, like night shift work. OSD processes have recently attracted attention in relation to time-resolved external stressor trajectories in personalized medicine (prevention) initiatives, as they seem to interact with circadian clock systems towards the improved delineation of the early stages of (chronic) disease process. OBJECTIVES: This work critically reviewed human studies targeting the temporal dynamics of OSD and circadian clock system's activity in response to environmental/occupational stressors; the case of night shift work was examined. METHODS: Being a key stressor influencing OSD processes and circadian rhythm, night shift work was evaluated as part of a scoping review of research in OSD, including inflammatory and metabolic processes to determine the extent of OSD research undertaken in human populations, methodologies, tools and biomarkers used and the extent that the temporal dimensions of exposure and biological effect(s) were accounted for. Online databases were searched for papers published from 2000 onwards, resulting in the selection of 53 original publications. RESULTS AND DISCUSSION: The majority of studies (n = 41) took place in occupational settings, while the rest were conducted in the general population or patient groups. Most occupational studies targeted outcomes of oxidative stress/damage (n = 19), followed by the combination of OSD with inflammatory response (n = 10), and studies focused on metabolic outcomes (n = 12). Only a minor fraction of the studies measured biomarkers related to circadian rhythm, such as, melatonin, its metabolite, or cortisol. Night shift work was associated with select biomarkers of OSD and inflammation, albeit with mixed results. Although much progress in delineating the biological mechanisms of OSD process has been made, an equally thorough investigation on the temporal trajectory of OSD processes as triggered by environmental/occupational stressors in human studies has yet to fully evolve.

Development of Erf-Mediated Craniosynostosis and Pharmacological Amelioration.

ETS2 repressor factor (ERF) insufficiency causes craniosynostosis (CRS4) in humans and mice. ERF is an ETS domain transcriptional repressor regulated by Erk1/2 phosphorylation via nucleo-cytoplasmic shuttling. Here, we analyze the onset and development of the craniosynostosis phenotype in an Erf-insufficient mouse model and evaluate the potential of the residual Erf activity augmented by pharmacological compounds to ameliorate the disease. Erf insufficiency appears to cause an initially compromised frontal bone formation and subsequent multisuture synostosis, reflecting distinct roles of Erf on the cells that give rise to skull and facial bones. We treated animals with Mek1/2 and nuclear export inhibitors, U0126 and KPT-330, respectively, to increase Erf activity by two independent pathways. We implemented both a low dosage locally over the calvaria and a systemic drug administration scheme to evaluate the possible indirect effects from other systems and minimize toxicity. The treatment of mice with either the inhibitors or the administration scheme alleviated the synostosis phenotype with minimal adverse effects. Our data suggest that the ERF level is an important regulator of cranial bone development and that pharmacological modulation of its activity may represent a valid intervention approach both in CRS4 and in other syndromic forms of craniosynostosis mediated by the FGFR-RAS-ERK-ERF pathway.

Thermalization of the Ablowitz-Ladik lattice in the presence of non-integrable perturbations.

We investigate the statistical mechanics of the photonic Ablowitz-Ladik lattice, the integrable version of the discrete nonlinear Schrödinger equation. In this regard, we demonstrate that in the presence of perturbations, the complex response of this system can be accurately captured within the framework of optical thermodynamics. Along these lines, we shed light on the true relevance of chaos in the thermalization of the Ablowitz-Ladik system. Our results indicate that when linear and nonlinear perturbations are incorporated, this weakly nonlinear lattice will thermalize into a proper Rayleigh-Jeans distribution with a well-defined temperature and chemical potential, in spite of the fact that the underlying nonlinearity is non-local and hence does not have a multi-wave mixing representation. This result illustrates that in the supermode basis, a non-local and non-Hermitian nonlinearity can in fact properly thermalize this periodic array in the presence of two quasi-conserved quantities.

EJE Prize 2023: genes on steroids-genomic control of hepatic metabolism by the glucocorticoid receptor.

Glucocorticoids are essential hormones produced by the adrenal cortex with prominent circadian rhythmicity and in times of stress. Glucocorticoids maintain liver homeostasis through coordinated activities that control the major pathways of energy metabolism. Glucocorticoids activate the glucocorticoid receptor (GR), a nuclear hormone receptor that regulates the transcription of hundreds of genes in response to ligand. This review aims to provide a comprehensive overview of glucocorticoid receptor signaling impact on glucose, amino acid, and lipid metabolism in the liver. We integrate fundamental and current findings elucidating key GR-regulated pathways from a physiologic, biochemical, and molecular point-of-view. Here, we focus on the transcriptional regulation of well-characterized hepatic GR target genes, and on those GR co-factors that coordinate nutritional and hormonal signals.

Sarcopenia in Rheumatoid arthritis. A narrative review.

Sarcopenia was recently identified as an entity in the ICD-10 classification of October 2016. According to the recommendation of the European Working Group on Sarcopenia in Older People (EWGSOP2), sarcopenia is defined as low muscle strength and low muscle mass, while physical performance is used to categorize the severity of sarcopenia. In recent years, sarcopenia has become increasingly common in younger patients with autoimmune diseases such as Rheumatoid arthritis (RA). Due to the chronic inflammation caused by RA, patients have reduced physical activity, immobility, stiffness, and joint destruction and all of that lead to the loss of muscle mass, muscle strength, disability and significantly lowering the patients' quality of life. This article is a narrative review about sarcopenia in RA, with a special focus in its pathogenesis and management.

Analytical performance specifications for the measurement uncertainty of 24,25-dihydroxyvitamin D examinations.

OBJECTIVES: The exploration of the metabolites in the degradation pathways of vitamin D (VTD) has gained importance in recent years and simultaneous quantitation of twenty-five-hydroxy vitamin D (25(OH)D) mass concentration together with 24,25-dihydroxyvitamin D (24,25(OH)2D) has been proposed as a newer approach to define VTD deficiency. Yet, no data are available on 24,25(OH)2D biological variation (BV). In this study, we evaluated 24,25(OH)2D's BV on the European Biological Variation Study (EuBIVAS) cohort samples to determine if analytical performance specifications (APS) for 24,25(OH)2D could be generated. METHODS: Six European laboratories recruited 91 healthy participants. 25(OH)D and 24,25(OH)2D concentrations in K3-EDTA plasma were examined weekly for up to 10 weeks in duplicate with a validated LC-MS/MS method. The Vitamin D Metabolite Ratio (24,25(OH)2D divided by 25(OH)D × 100) was also calculated at each time point. RESULTS: Linear regression of the mean 24,25(OH)2D concentrations at each blood collection showed participants were not in steady state. Variations of 24,25(OH)2D over time were significantly positively associated with the slopes of 25(OH)D concentrations over time and the concentration of 25(OH)D of the participant at inclusion, and negatively associated with body mass index (BMI), but not with age, gender, or location of the participant. The variation of the 24,25(OH)2D concentration in participants over a 10 weeks period was 34.6%. Methods that would detect a significant change linked to the natural production of 24,25(OH)2D over this period at p<0.05 would need a relative measurement uncertainty (u%)<14.9% while at p<0.01, relative measurement uncertainty should be <10.5%. CONCLUSIONS: We have defined for the first time APS for 24,25(OH)2D examinations. According to the growing interest in this metabolite, several laboratories and manufacturers might aim to develop specific methods for its determination. The results presented in this paper are thus necessary prerequisites for the validation of such methods.