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Reducing foodborne disease incidence is a public health priority. This report summarizes preliminary 2023 Foodborne Diseases Active Surveillance Network (FoodNet) data and highlights efforts to increase the representativeness of FoodNet. During 2023, incidences of domestically acquired campylobacteriosis, Shiga toxin-producing Escherichia coli infection, yersiniosis, vibriosis, and cyclosporiasis increased, whereas those of listeriosis, salmonellosis, and shigellosis remained stable compared with incidences during 2016-2018, the baseline used for tracking progress towards federal disease reduction goals. During 2023, the incidence and percentage of infections diagnosed by culture-independent diagnostic tests (CIDTs) reported to FoodNet continued to increase, and the percentage of cases that yielded an isolate decreased, affecting observed trends in incidence. Because CIDTs allow for diagnosis of infections that previously would have gone undetected, lack of progress toward disease reduction goals might reflect changing diagnostic practices rather than an actual increase in incidence. Continued surveillance is needed to monitor the impact of changing diagnostic practices on disease trends, and targeted prevention efforts are needed to meet disease reduction goals. During 2023, FoodNet expanded its catchment area for the first time since 2004. This expansion improved the representativeness of the FoodNet catchment area, the ability of FoodNet to monitor trends in disease incidence, and the generalizability of FoodNet data., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Tamara Rissman reports that she is an adjunct professor in the Public Health Department at Southern Connecticut State University. No other potential conflicts of interest were disclosed.

Metabolomic age models have been proposed for the study of biological aging, however, they have not been widely validated. We aimed to assess the performance of newly developed and existing nuclear magnetic resonance spectroscopy (NMR) metabolomic age models for prediction of chronological age (CA), mortality, and age-related disease. Ninety-eight metabolic variables were measured in blood from nine UK and Finnish cohort studies (N ≈31,000 individuals, age range 24-86 years). We used nonlinear and penalized regression to model CA and time to all-cause mortality. We examined associations of four new and two previously published metabolomic age models, with aging risk factors and phenotypes. Within the UK Biobank (N ≈102,000), we tested prediction of CA, incident disease (cardiovascular disease (CVD), type-2 diabetes mellitus, cancer, dementia, and chronic obstructive pulmonary disease), and all-cause mortality. Seven-fold cross-validated Pearson's r between metabolomic age models and CA ranged between 0.47 and 0.65 in the training cohort set (mean absolute error: 8-9 years). Metabolomic age models, adjusted for CA, were associated with C-reactive protein, and inversely associated with glomerular filtration rate. Positively associated risk factors included obesity, diabetes, smoking, and physical inactivity. In UK Biobank, correlations of metabolomic age with CA were modest (r = 0.29-0.33), yet all metabolomic model scores predicted mortality (hazard ratios of 1.01 to 1.06/metabolomic age year) and CVD, after adjustment for CA. While metabolomic age models were only moderately associated with CA in an independent population, they provided additional prediction of morbidity and mortality over CA itself, suggesting their wider applicability., (© 2024 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Metabolomics, the global profiling of small molecules in the body, has emerged as a promising analytical approach for assessing molecular changes associated with ageing at the population level. Understanding root metabolic ageing pathways may have important implications for managing age-related disease risk. In this short review, relevant studies published in the last few years that have made valuable contributions to this field will be discussed. These include large-scale studies investigating metabolic changes with age, metabolomic clocks, and metabolic pathways associated with ageing phenotypes. Recent significant advances include the use of longitudinal study designs, populations spanning the whole life course, standardised analytical platforms of enhanced metabolome coverage and development of multivariate analyses. While many challenges remain, recent studies have demonstrated the considerable promise of this field., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Oliver Robinson reports financial support was provided by UK Research and Innovation., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Each year, infections from major foodborne pathogens are responsible for an estimated 9.4 million illnesses, 56,000 hospitalizations, and 1,350 deaths in the United States (1). To evaluate progress toward prevention of enteric infections in the United States, the Foodborne Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-diagnosed infections caused by eight pathogens transmitted commonly through food at 10 U.S. sites. During 2020-2021, FoodNet detected decreases in many infections that were due to behavioral modifications, public health interventions, and changes in health care-seeking and testing practices during the COVID-19 pandemic. This report presents preliminary estimates of pathogen-specific annual incidences during 2022, compared with average annual incidences during 2016-2018, the reference period for the U.S. Department of Health and Human Services' Healthy People 2030 targets (2). Many pandemic interventions ended by 2022, resulting in a resumption of outbreaks, international travel, and other factors leading to enteric infections. During 2022, annual incidences of illnesses caused by the pathogens Campylobacter, Salmonella, Shigella, and Listeria were similar to average annual incidences during 2016-2018; however, incidences of Shiga toxin-producing Escherichia coli (STEC), Yersinia, Vibrio, and Cyclospora illnesses were higher. Increasing culture-independent diagnostic test (CIDT) usage likely contributed to increased detection by identifying infections that would have remained undetected before widespread CIDT usage. Reducing pathogen contamination during poultry slaughter and processing of leafy greens requires collaboration among food growers and processors, retail stores, restaurants, and regulators., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. Elaine Scallan Walter reports consulting fees for a peer review of a Salmonella risk profile for the U.S. Department of Agriculture Food Safety and Inspection Service via RTI International during 2022. No other potential conflicts of interest were disclosed.

Background: While biological age in adults is often understood as representing general health and resilience, the conceptual interpretation of accelerated biological age in children and its relationship to development remains unclear. We aimed to clarify the relationship of accelerated biological age, assessed through two established biological age indicators, telomere length and DNA methylation age, and two novel candidate biological age indicators, to child developmental outcomes, including growth and adiposity, cognition, behavior, lung function and the onset of puberty, among European school-age children participating in the HELIX exposome cohort., Methods: The study population included up to 1173 children, aged between 5 and 12 years, from study centres in the UK, France, Spain, Norway, Lithuania, and Greece. Telomere length was measured through qPCR, blood DNA methylation, and gene expression was measured using microarray, and proteins and metabolites were measured by a range of targeted assays. DNA methylation age was assessed using Horvath's skin and blood clock, while novel blood transcriptome and 'immunometabolic' (based on plasma proteins and urinary and serum metabolites) clocks were derived and tested in a subset of children assessed six months after the main follow-up visit. Associations between biological age indicators with child developmental measures as well as health risk factors were estimated using linear regression, adjusted for chronological age, sex, ethnicity, and study centre. The clock derived markers were expressed as Δ age (i.e. predicted minus chronological age)., Results: Transcriptome and immunometabolic clocks predicted chronological age well in the test set ( r =0.93 and r =0.84 respectively). Generally, weak correlations were observed, after adjustment for chronological age, between the biological age indicators.Among associations with health risk factors, higher birthweight was associated with greater immunometabolic Δ age, smoke exposure with greater DNA methylation Δ age, and high family affluence with longer telomere length.Among associations with child developmental measures, all biological age markers were associated with greater BMI and fat mass, and all markers except telomere length were associated with greater height, at least at nominal significance (p<0.05). Immunometabolic Δ age was associated with better working memory (p=4 e-3) and reduced inattentiveness (p=4 e-4), while DNA methylation Δ age was associated with greater inattentiveness (p=0.03) and poorer externalizing behaviors (p=0.01). Shorter telomere length was also associated with poorer externalizing behaviors (p=0.03)., Conclusions: In children, as in adults, biological aging appears to be a multi-faceted process and adiposity is an important correlate of accelerated biological aging. Patterns of associations suggested that accelerated immunometabolic age may be beneficial for some aspects of child development while accelerated DNA methylation age and telomere attrition may reflect early detrimental aspects of biological aging, apparent even in children., Funding: UK Research and Innovation (MR/S03532X/1); European Commission (grant agreement numbers: 308333; 874583)., Competing Interests: OR, CL, SJ, SA, EB, Pd, LC, HK, RG, KG, LM, DM, ES, VS, JU, MV, JW, TN, MB, MV No competing interests declared, (© 2023, Robinson et al.)

Background: Individuals are exposed to environmental pollutants with endocrine disrupting activity (endocrine disruptors, EDCs) and the early stages of life are particularly susceptible to these exposures. Previous studies have focused on identifying molecular signatures associated with EDCs, but none have used repeated sampling strategy and integrated multiple omics. We aimed to identify multi-omic signatures associated with childhood exposure to non-persistent EDCs., Methods: We used data from the HELIX Child Panel Study, which included 156 children aged 6 to 11. Children were followed for one week, in two time periods. Twenty-two non-persistent EDCs (10 phthalate, 7 phenol, and 5 organophosphate pesticide metabolites) were measured in two weekly pools of 15 urine samples each. Multi-omic profiles (methylome, serum and urinary metabolome, proteome) were measured in blood and in a pool urine samples. We developed visit-specific Gaussian Graphical Models based on pairwise partial correlations. The visit-specific networks were then merged to identify reproducible associations. Independent biological evidence was systematically sought to confirm some of these associations and assess their potential health implications., Results: 950 reproducible associations were found among which 23 were direct associations between EDCs and omics. For 9 of them, we were able to find corroborating evidence from previous literature: DEP - serotonin, OXBE - cg27466129, OXBE - dimethylamine, triclosan - leptin, triclosan - serotonin, MBzP - Neu5AC, MEHP - cg20080548, oh-MiNP - kynurenine, oxo-MiNP - 5-oxoproline. We used these associations to explore possible mechanisms between EDCs and health outcomes, and found links to health outcomes for 3 analytes: serotonin and kynurenine in relation to neuro-behavioural development, and leptin in relation to obesity and insulin resistance., Conclusions: This multi-omics network analysis at two time points identified biologically relevant molecular signatures related to non-persistent EDC exposure in childhood, suggesting pathways related to neurological and metabolic outcomes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Environmental exposures during early life play a critical role in life-course health, yet the molecular phenotypes underlying environmental effects on health are poorly understood. In the Human Early Life Exposome (HELIX) project, a multi-centre cohort of 1301 mother-child pairs, we associate individual exposomes consisting of >100 chemical, outdoor, social and lifestyle exposures assessed in pregnancy and childhood, with multi-omics profiles (methylome, transcriptome, proteins and metabolites) in childhood. We identify 1170 associations, 249 in pregnancy and 921 in childhood, which reveal potential biological responses and sources of exposure. Pregnancy exposures, including maternal smoking, cadmium and molybdenum, are predominantly associated with child DNA methylation changes. In contrast, childhood exposures are associated with features across all omics layers, most frequently the serum metabolome, revealing signatures for diet, toxic chemical compounds, essential trace elements, and weather conditions, among others. Our comprehensive and unique resource of all associations ( https://helixomics.isglobal.org/ ) will serve to guide future investigation into the biological imprints of the early life exposome., (© 2022. The Author(s).)

Background: Adherence to the Dietary Approaches to Stop Hypertension (DASH) diet enhances potassium intake and reduces sodium intake and blood pressure (BP), but the underlying metabolic pathways are unclear., Objectives: Among free-living populations, we delineated metabolic signatures associated with the DASH diet adherence, 24-hour urinary sodium and potassium excretions, and the potential metabolic pathways involved., Methods: We used 24-hour urinary metabolic profiling by proton nuclear magnetic resonance spectroscopy to characterize the metabolic signatures associated with the DASH dietary pattern score (DASH score) and 24-hour excretion of sodium and potassium among participants in the United States (n = 2164) and United Kingdom (n = 496) enrolled in the International Study of Macro- and Micronutrients and Blood Pressure (INTERMAP). Multiple linear regression and cross-tabulation analyses were used to investigate the DASH-BP relation and its modulation by sodium and potassium. Potential pathways associated with DASH adherence, sodium and potassium excretion, and BP were identified using mediation analyses and metabolic reaction networks., Results: Adherence to the DASH diet was associated with urinary potassium excretion (correlation coefficient, r = 0.42; P < 0.0001). In multivariable regression analyses, a 5-point higher DASH score (range, 7 to 35) was associated with a lower systolic BP by 1.35 mmHg (95% CI, -1.95 to -0.80 mmHg; P = 1.2 × 10-5); control of the model for potassium but not sodium attenuated the DASH-BP relation. Two common metabolites (hippurate and citrate) mediated the potassium-BP and DASH-BP relationships, while 5 metabolites (succinate, alanine, S-methyl cysteine sulfoxide, 4-hydroxyhippurate, and phenylacetylglutamine) were found to be specific to the DASH-BP relation., Conclusions: Greater adherence to the DASH diet is associated with lower BP and higher potassium intake across levels of sodium intake. The DASH diet recommends greater intake of fruits, vegetables, and other potassium-rich foods that may replace sodium-rich processed foods and thereby influence BP through overlapping metabolic pathways. Possible DASH-specific pathways are speculated but confirmation requires further study. INTERMAP is registered as NCT00005271 at www.clinicaltrials.gov., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society for Nutrition.)

Untargeted metabolomics and lipidomics LC-MS experiments produce complex datasets, usually containing tens of thousands of features from thousands of metabolites whose annotation requires additional MS/MS experiments and expert knowledge. All-ion fragmentation (AIF) LC-MS/MS acquisition provides fragmentation data at no additional experimental time cost. However, analysis of such datasets requires reconstruction of parent-fragment relationships and annotation of the resulting pseudo-MS/MS spectra. Here, we propose a novel approach for automated annotation of isotopologues, adducts, and in-source fragments from AIF LC-MS datasets by combining correlation-based parent-fragment linking with molecular fragment matching. Our workflow focuses on a subset of features rather than trying to annotate the full dataset, saving time and simplifying the process. We demonstrate the workflow in three human serum datasets containing 599 features manually annotated by experts. Precision and recall values of 82-92% and 82-85%, respectively, were obtained for features found in the highest-rank scores (1-5). These results equal or outperform those obtained using MS-DIAL software, the current state of the art for AIF data annotation. Further validation for other biological matrices and different instrument types showed variable precision (60-89%) and recall (10-88%) particularly for datasets dominated by nonlipid metabolites. The workflow is freely available as an open-source R package, MetaboAnnotatoR, together with the fragment libraries from Github (https://github.com/gggraca/MetaboAnnotatoR).

Urinary metabolic profiling is a promising powerful tool to reflect dietary intake and can help understand metabolic alterations in response to diet quality. Here, we used 1 H NMR spectroscopy in a multicountry study in European children (1147 children from 6 different cohorts) and identified a common panel of 4 urinary metabolites (hippurate, N -methylnicotinic acid, urea, and sucrose) that was predictive of Mediterranean diet adherence (KIDMED) and ultra-processed food consumption and also had higher capacity in discriminating children's diet quality than that of established sociodemographic determinants. Further, we showed that the identified metabolite panel also reflected the associations of these diet quality indicators with C-peptide, a stable and accurate marker of insulin resistance and future risk of metabolic disease. This methodology enables objective assessment of dietary patterns in European child populations, complementary to traditional questionary methods, and can be used in future studies to evaluate diet quality. Moreover, this knowledge can provide mechanistic evidence of common biological pathways that characterize healthy and unhealthy dietary patterns, and diet-related molecular alterations that could associate to metabolic disease., Competing Interests: NS, AS, EP, AN, YZ, KM, CL, LM, SF, LA, SA, XB, AB, MC, SF, RG, BH, RM, HM, CM, FR, OR, TR, EB, ES, JU, MV, PV, TV, JW, DC, MV, HK, LC No competing interests declared, MC Is an employee of AstraZeneca. The author declares that no other competing interests exist, (© 2022, Stratakis et al.)

Background: Fibrinogen (FIB) levels less than 150 mg/dL have been associated with increased rates of bleeding and lower survival in critically ill cirrhosis patients., Objective: We aimed to determine if treatment with cryoprecipitate (CRYO) for low FIB levels is associated with bleeding outcomes or survival., Methods: A total of 237 cirrhosis patients admitted to an intensive care unit at a tertiary care liver transplant center with initial FIB levels less than 150 mg/dL were retrospectively assessed for CRYO transfusion, bleeding events, and survival outcomes., Results: The mean MELD score was 27.2 (95% confidence interval [CI]: 26.0-28.3) and CLIF-C acute on chronic liver failure score was 53.4 (51.9-54.8). Ninety-nine (41.8%) were admitted for acute bleeding and the remainder were admitted for nonbleeding illnesses. FIB level on admission correlated strongly with disease severity. After adjusting for disease severity, FIB on admission was not an independent predictor of 30-day survival (hazard ratio [HR]: 0.99, 95% CI: 0.99-1.01, p  = 0.68). CRYO transfusion increased FIB levels but had no independent effect on mortality or bleeding complications (HR: 1.10, 95% CI: 0.72-1.70, p  = 0.65)., Conclusion: In cirrhosis patients with critical illness, low FIB levels on presentation reflect severity of illness but are not independently associated with 30-day mortality. Treatment of low FIB with CRYO also does not affect survival or bleeding complications, suggesting FIB is an additional marker of severity of illness but is not itself a direct factor in the pathophysiology of bleeding in critically ill cirrhosis patients., Competing Interests: None declared., (Thieme. All rights reserved.)

Background: Multiple omics technologies are increasingly applied to detect early, subtle molecular responses to environmental stressors for future disease risk prevention. However, there is an urgent need for further evaluation of stability and variability of omics profiles in healthy individuals, especially during childhood., Methods: We aimed to estimate intra-, inter-individual and cohort variability of multi-omics profiles (blood DNA methylation, gene expression, miRNA, proteins and serum and urine metabolites) measured 6 months apart in 156 healthy children from five European countries. We further performed a multi-omics network analysis to establish clusters of co-varying omics features and assessed the contribution of key variables (including biological traits and sample collection parameters) to omics variability., Results: All omics displayed a large range of intra- and inter-individual variability depending on each omics feature, although all presented a highest median intra-individual variability. DNA methylation was the most stable profile (median 37.6% inter-individual variability) while gene expression was the least stable (6.6%). Among the least stable features, we identified 1% cross-omics co-variation between CpGs and metabolites (e.g. glucose and CpGs related to obesity and type 2 diabetes). Explanatory variables, including age and body mass index (BMI), explained up to 9% of serum metabolite variability., Conclusions: Methylation and targeted serum metabolomics are the most reliable omics to implement in single time-point measurements in large cross-sectional studies. In the case of metabolomics, sample collection and individual traits (e.g. BMI) are important parameters to control for improved comparability, at the study design or analysis stage. This study will be valuable for the design and interpretation of epidemiological studies that aim to link omics signatures to disease, environmental exposures, or both., (© 2021. The Author(s).)

Background: Low socio-economic position (SEP) is a risk factor for multiple health outcomes, but its molecular imprints in the body remain unclear., Methods: We examined SEP as a determinant of serum nuclear magnetic resonance metabolic profiles in ∼30 000 adults and 4000 children across 10 UK and Finnish cohort studies., Results: In risk-factor-adjusted analysis of 233 metabolic measures, low educational attainment was associated with 37 measures including higher levels of triglycerides in small high-density lipoproteins (HDL) and lower levels of docosahexaenoic acid (DHA), omega-3 fatty acids, apolipoprotein A1, large and very large HDL particles (including levels of their respective lipid constituents) and cholesterol measures across different density lipoproteins. Among adults whose father worked in manual occupations, associations with apolipoprotein A1, large and very large HDL particles and HDL-2 cholesterol remained after adjustment for SEP in later life. Among manual workers, levels of glutamine were higher compared with non-manual workers. All three indicators of low SEP were associated with lower DHA, omega-3 fatty acids and HDL diameter. At all ages, children of manual workers had lower levels of DHA as a proportion of total fatty acids., Conclusions: Our work indicates that social and economic factors have a measurable impact on human physiology. Lower SEP was independently associated with a generally unfavourable metabolic profile, consistent across ages and cohorts. The metabolites we found to be associated with SEP, including DHA, are known to predict cardiovascular disease and cognitive decline in later life and may contribute to health inequalities., (© The Author(s) 2020. Published by Oxford University Press on behalf of the International Epidemiological Association.)

Cancer is well established as an age-associated disease, and there is substantial overlap in the molecular, cellular and physiological changes observed with both ageing and the progression of cancer. Age-specific declines in resilience mechanisms such as DNA repair or epigenetic maintenance may contribute to the development of cancer. These declines may be assessed through biomarkers that measure biological age and through the related concept of "age acceleration". Epigenetic clocks, assessed through DNA methylation levels, are among the most widely used biological age markers in cancer studies. In this review, we discuss the use of DNA methylation ageing measures to predict population cancer incidence, mortality and survival. Blood-based DNA methylation age estimators appear to be promising measures of increased cancer risk and mortality, although their reported effects are generally weak, thus its clinical relevance remains to be validated in large case-cohort and longitudinal studies. Future development of epigenetic and other biological age biomarkers will likely further elucidate the links between ageing and cancer., (© 2020 UICC.)

Human metabolism is influenced by genetic and environmental factors. Previous studies have identified over 23 loci associated with more than 26 urine metabolites levels in adults, which are known as urinary metabolite quantitative trait loci (metabQTLs). The aim of the present study is the identification for the first time of urinary metabQTLs in children and their interaction with dietary patterns. Association between genome-wide genotyping data and 44 urine metabolite levels measured by proton nuclear magnetic resonance spectroscopy was tested in 996 children from the Human Early Life Exposome project. Twelve statistically significant urine metabQTLs were identified, involving 11 unique loci and 10 different metabolites. Comparison with previous findings in adults revealed that six metabQTLs were already known, and one had been described in serum and three were involved the same locus as other reported metabQTLs but had different urinary metabolites. The remaining two metabQTLs represent novel urine metabolite-locus associations, which are reported for the first time in this study [single nucleotide polymorphism (SNP) rs12575496 for taurine, and the missense SNP rs2274870 for 3-hydroxyisobutyrate]. Moreover, it was found that urinary taurine levels were affected by the combined action of genetic variation and dietary patterns of meat intake as well as by the interaction of this SNP with beverage intake dietary patterns. Overall, we identified 12 urinary metabQTLs in children, including two novel associations. While a substantial part of the identified loci affected urinary metabolite levels both in children and in adults, the metabQTL for taurine seemed to be specific to children and interacted with dietary patterns., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: Overweight and obesity amongst women of reproductive age are increasingly common in developed economies and are shown to adversely affect birth outcomes and both childhood and adulthood health risks in the offspring. Metabolic profiling in conditions of overweight and obesity in pregnancy could potentially be applied to elucidate the molecular basis of the adverse effects of gestational weight gain (GWG) and postpartum weight loss (WL) on future risks for cardiovascular disease (CVD) and other chronic diseases., Methods: Biofluid samples were collected from 114 ethnically diverse pregnant women with body mass index (BMI) between 25 and 40 kg/m 2 from Chicago (US), as part of a randomized lifestyle intervention trial (Maternal Offspring Metabolics: Family Intervention Trial; NCT01631747). At 15 weeks, 35 weeks of gestation, and at 1 year postpartum, the blood plasma lipidome and metabolic profile of urine samples were analyzed by liquid chromatography mass spectrometry (LC-MS) and 1 H nuclear magnetic resonance spectroscopy ( 1 H NMR) respectively., Results: Urinary 4-deoxyerythronic acid and 4-deoxythreonic acid were found to be positively correlated to BMI. Seventeen plasma lipids were found to be associated with GWG and 16 lipids were found to be associated with WL, which included phosphatidylinositols (PI), phosphatidylcholines (PC), lysophospholipids (lyso-), sphingomyelins (SM) and ether phosphatidylcholine (PC-O). Three phospholipids found to be positively associated with GWG all contained palmitate side-chains, and amongst the 14 lipids that were negatively associated with GWG, seven were PC-O. Six of eight lipids found to be negatively associated with WL contained an 18:2 fatty acid side-chain., Conclusions: Maternal obesity was associated with characteristic urine and plasma metabolic phenotypes, and phospholipid profile was found to be associated with both GWG and postpartum WL in metabolically healthy pregnant women with overweight/obesity. Postpartum WL may be linked to the reduction in the intake of linoleic acid/conjugated linoleic acid food sources in our study population.

Background: The adverse health effects of early life exposure to tobacco smoking have been widely reported. In spite of this, the underlying molecular mechanisms of in utero and postnatal exposure to tobacco smoke are only partially understood. Here, we aimed to identify multi-layer molecular signatures associated with exposure to tobacco smoke in these two exposure windows., Methods: We investigated the associations of maternal smoking during pregnancy and childhood secondhand smoke (SHS) exposure with molecular features measured in 1203 European children (mean age 8.1 years) from the Human Early Life Exposome (HELIX) project. Molecular features, covering 4 layers, included blood DNA methylation and gene and miRNA transcription, plasma proteins, and sera and urinary metabolites., Results: Maternal smoking during pregnancy was associated with DNA methylation changes at 18 loci in child blood. DNA methylation at 5 of these loci was related to expression of the nearby genes. However, the expression of these genes themselves was only weakly associated with maternal smoking. Conversely, childhood SHS was not associated with blood DNA methylation or transcription patterns, but with reduced levels of several serum metabolites and with increased plasma PAI1 (plasminogen activator inhibitor-1), a protein that inhibits fibrinolysis. Some of the in utero and childhood smoking-related molecular marks showed dose-response trends, with stronger effects with higher dose or longer duration of the exposure., Conclusion: In this first study covering multi-layer molecular features, pregnancy and childhood exposure to tobacco smoke were associated with distinct molecular phenotypes in children. The persistent and dose-dependent changes in the methylome make CpGs good candidates to develop biomarkers of past exposure. Moreover, compared to methylation, the weak association of maternal smoking in pregnancy with gene expression suggests different reversal rates and a methylation-based memory to past exposures. Finally, certain metabolites and protein markers evidenced potential early biological effects of postnatal SHS, such as fibrinolysis.

Identification of genomic and epigenomic determinants of drug resistance provides important insights for improving cancer treatment. Using agnostic genome-wide interrogation of mRNA and miRNA expression, DNA methylation, SNPs, CNAs and SNVs/Indels in primary human acute lymphoblastic leukemia cells, we identified 463 genomic features associated with glucocorticoid resistance. Gene-level aggregation identified 118 overlapping genes, 15 of which were confirmed by genome-wide CRISPR screen. Collectively, this identified 30 of 38 (79%) known glucocorticoid-resistance genes/miRNAs and all 38 known resistance pathways, while revealing 14 genes not previously associated with glucocorticoid-resistance. Single cell RNAseq and network-based transcriptomic modelling corroborated the top previously undiscovered gene, CELSR2. Manipulation of CELSR2 recapitulated glucocorticoid resistance in human leukemia cell lines and revealed a synergistic drug combination (prednisolone and venetoclax) that mitigated resistance in mouse xenograft models. These findings illustrate the power of an integrative genomic strategy for elucidating genes and pathways conferring drug resistance in cancer cells., Competing Interests: Competing Interests The authors declare no competing interests.

Background: Results from observational studies regarding associations between fish (including shellfish) intake and cardiovascular disease risk factors, including blood pressure (BP) and BMI, are inconsistent., Objective: To investigate associations of fish consumption and associated urinary metabolites with BP and BMI in free-living populations., Methods: We used cross-sectional data from the International Study of Macro-/Micronutrients and Blood Pressure (INTERMAP), including 4680 men and women (40-59 y) from Japan, China, the United Kingdom, and United States. Dietary intakes were assessed by four 24-h dietary recalls and BP from 8 measurements. Urinary metabolites (2 timed 24-h urinary samples) associated with fish intake acquired from NMR spectroscopy were identified. Linear models were used to estimate BP and BMI differences across categories of intake and per 2 SD higher intake of fish and its biomarkers., Results: No significant associations were observed between fish intake and BP. There was a direct association with fish intake and BMI in the Japanese population sample (P trend = 0.03; fully adjusted model). In Japan, trimethylamine-N-oxide (TMAO) and taurine, respectively, demonstrated area under the receiver operating characteristic curve (AUC) values of 0.81 and 0.78 in discriminating high against low fish intake, whereas homarine (a metabolite found in shellfish muscle) demonstrated an AUC of 0.80 for high/nonshellfish intake. Direct associations were observed between urinary TMAO and BMI for all regions except Japan (P < 0.0001) and in Western populations between TMAO and BP (diastolic blood pressure: mean difference 1.28; 95% CI: 0.55, 2.02 mmHg; P = 0.0006, systolic blood pressure: mean difference 1.67; 95% CI: 0.60, 2.73 mmHg; P = 0.002)., Conclusions: Urinary TMAO showed a stronger association with fish intake in the Japanese compared with the Western population sample. Urinary TMAO was directly associated with BP in the Western but not the Japanese population sample. Associations between fish intake and its biomarkers and downstream associations with BP/BMI appear to be context specific. INTERMAP is registered at www.clinicaltrials.gov as NCT00005271., (Copyright © The Author(s) 2019.)

Altered metabolism is one of the key hallmarks of cancer. The development of sensitive, reproducible and robust bioanalytical tools such as Nuclear Magnetic Resonance Spectroscopy and Mass Spectrometry techniques offers numerous opportunities for cancer metabolism research, and provides additional and exciting avenues in cancer diagnosis, prognosis and for the development of more effective and personalized treatments. In this chapter, we introduce the current state of the art of metabolomics and metabolic phenotyping approaches in cancer research and clinical diagnostics.

Objective: The ratio of the partial pressure of arterial oxygen to fraction of inspired oxygen is a key component of the sequential organ failure assessment score that operationally defines sepsis. But, it is calculated infrequently due to the need for the acquisition of an arterial blood gas. So, we sought to find an optimal imputation strategy for the estimation of sepsis-defining hypoxemic respiratory failure using oximetry instead of an arterial blood gas., Approach: We retrospectively studied a sample of non-intubated acute-care patients with oxygen saturation recorded  ⩽10 min before arterial blood sampling (N  =  492 from 2013-2017). We imputed ratios of the partial pressure of arterial oxygen to the fraction of inspired oxygen and sepsis criteria from existing imputation equations (Hill, Severinghaus-Ellis, Rice, and Pandharipande) and compared them with the ratios and sepsis criteria measured from arterial blood gases. We devised a modified model-based equation to eliminate the bias of the results., Main Results: Hypoxemia severity estimates from the Severinghaus-Ellis equation were more accurate than those from other existing equations, but showed significant proportional bias towards under-estimation of hypoxemia severity, especially at oxygen saturations  >96%. Our modified equation eliminated bias and surpassed others on all imputation quality metrics., Significance: Our modified imputation equation, [Formula: see text] is the first one that is free of bias at all oxygen saturations. It resulted in ratios of partial pressure of arterial oxygen to fraction of inspired oxygen and sepsis respiratory criteria closest to those obtained by arterial blood gas testing and is the optimal imputation strategy for non-intubated acute-care patients.

Background: Environment and diet in early life can affect development and health throughout the life course. Metabolic phenotyping of urine and serum represents a complementary systems-wide approach to elucidate environment-health interactions. However, large-scale metabolome studies in children combining analyses of these biological fluids are lacking. Here, we sought to characterise the major determinants of the child metabolome and to define metabolite associations with age, sex, BMI and dietary habits in European children, by exploiting a unique biobank established as part of the Human Early-Life Exposome project ( http://www.projecthelix.eu )., Methods: Metabolic phenotypes of matched urine and serum samples from 1192 children (aged 6-11) recruited from birth cohorts in six European countries were measured using high-throughput 1 H nuclear magnetic resonance (NMR) spectroscopy and a targeted LC-MS/MS metabolomic assay (Biocrates AbsoluteIDQ p180 kit)., Results: We identified both urinary and serum creatinine to be positively associated with age. Metabolic associations to BMI z-score included a novel association with urinary 4-deoxyerythreonic acid in addition to valine, serum carnitine, short-chain acylcarnitines (C3, C5), glutamate, BCAAs, lysophosphatidylcholines (lysoPC a C14:0, lysoPC a C16:1, lysoPC a C18:1, lysoPC a C18:2) and sphingolipids (SM C16:0, SM C16:1, SM C18:1). Dietary-metabolite associations included urinary creatine and serum phosphatidylcholines (4) with meat intake, serum phosphatidylcholines (12) with fish, urinary hippurate with vegetables, and urinary proline betaine and hippurate with fruit intake. Population-specific variance (age, sex, BMI, ethnicity, dietary and country of origin) was better captured in the serum than in the urine profile; these factors explained a median of 9.0% variance amongst serum metabolites versus a median of 5.1% amongst urinary metabolites. Metabolic pathway correlations were identified, and concentrations of corresponding metabolites were significantly correlated (r > 0.18) between urine and serum., Conclusions: We have established a pan-European reference metabolome for urine and serum of healthy children and gathered critical resources not previously available for future investigations into the influence of the metabolome on child health. The six European cohort populations studied share common metabolic associations with age, sex, BMI z-score and main dietary habits. Furthermore, we have identified a novel metabolic association between threonine catabolism and BMI of children.

Purpose: Essential to exposome research is the collection of data on many environmental exposures from different domains in the same subjects. The aim of the Human Early Life Exposome (HELIX) study was to measure and describe multiple environmental exposures during early life (pregnancy and childhood) in a prospective cohort and associate these exposures with molecular omics signatures and child health outcomes. Here, we describe recruitment, measurements available and baseline data of the HELIX study populations., Participants: The HELIX study represents a collaborative project across six established and ongoing longitudinal population-based birth cohort studies in six European countries (France, Greece, Lithuania, Norway, Spain and the UK). HELIX used a multilevel study design with the entire study population totalling 31 472 mother-child pairs, recruited during pregnancy, in the six existing cohorts (first level); a subcohort of 1301 mother-child pairs where biomarkers, omics signatures and child health outcomes were measured at age 6-11 years (second level) and repeat-sampling panel studies with around 150 children and 150 pregnant women aimed at collecting personal exposure data (third level)., Findings to Date: Cohort data include urban environment, hazardous substances and lifestyle-related exposures for women during pregnancy and their offspring from birth until 6-11 years. Common, standardised protocols were used to collect biological samples, measure exposure biomarkers and omics signatures and assess child health across the six cohorts. Baseline data of the cohort show substantial variation in health outcomes and determinants between the six countries, for example, in family affluence levels, tobacco smoking, physical activity, dietary habits and prevalence of childhood obesity, asthma, allergies and attention deficit hyperactivity disorder., Future Plans: HELIX study results will inform on the early life exposome and its association with molecular omics signatures and child health outcomes. Cohort data are accessible for future research involving researchers external to the project., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2018. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The application of metabolic phenotyping in clinical and epidemiological studies is limited by a poor understanding of inter-individual, intra-individual and temporal variability in metabolic phenotypes. Using 1 H NMR spectroscopy we characterised short-term variability in urinary metabolites measured from 20 children aged 8-9 years old. Daily spot morning, night-time and pooled (50:50 morning and night-time) urine samples across six days (18 samples per child) were analysed, and 44 metabolites quantified. Intraclass correlation coefficients (ICC) and mixed effect models were applied to assess the reproducibility and biological variance of metabolic phenotypes. Excellent analytical reproducibility and precision was demonstrated for the 1 H NMR spectroscopic platform (median CV 7.2%). Pooled samples captured the best inter-individual variability with an ICC of 0.40 (median). Trimethylamine, N-acetyl neuraminic acid, 3-hydroxyisobutyrate, 3-hydroxybutyrate/3-aminoisobutyrate, tyrosine, valine and 3-hydroxyisovalerate exhibited the highest stability with over 50% of variance specific to the child. The pooled sample was shown to capture the most inter-individual variance in the metabolic phenotype, which is of importance for molecular epidemiology study design. A substantial proportion of the variation in the urinary metabolome of children is specific to the individual, underlining the potential of such data to inform clinical and exposome studies conducted early in life.

Somatic mutations in PIK3CA are frequently found in a number of human cancers, including breast cancer, altering cellular physiology and tumour sensitivity to chemotherapy. This renders PIK3CA an attractive molecular target for early detection and personalised therapy. Using 1 H Nuclear Magnetic Resonance spectroscopy (NMR) and Gas Chromatography - Mass Spectrometery (GC-MS) together with 13 C stable isotope-labelled glucose and glutamine as metabolic tracers, we probed the phenotypic changes in metabolism following a single copy knock-in of mutant PIK3CA (H1047R) in the MCF10A cell line, an important cell model for studying oncogenic transformation in breast tissues. We observed effects in several metabolic pathways, including a decrease in glycerophosphocholine level together with increases in glutaminolysis, de novo fatty acid synthesis and pyruvate entry into the tricarboxylic acid cycle. Our findings highlight altered glyceroplipid metabolism and lipogenesis, as key metabolic phenotypes of mutant PIK3CA transformation that are recapitulated in the MCF10A cellular model.

We characterized the effects of i.v. and s.c. zolpidem (1-8 mg/kg) under a differential reinforcement of low-rate schedule (i.e. DRL 45 s) in 3-hour sessions. Both behavioral and pharmacokinetic-pharmacodynamic analyses were used with the intent to compare the effects of zolpidem with those of benzodiazepines reported previously under the same behavioral paradigm. Zolpidem increased the shorter-response [inter-response times (IRTs)<45 s] rate and decreased the reinforcement rate in a dose- and time-related fashion. The behavioral profiles of zolpidem were mainly similar to those of benzodiazepines, except zolpidem produced far fewer shorter IRT responses. Pharmacokinetically, zolpidem decays biexponentially with distributional and terminal elimination half-lives of 5.2 and 42 min, respectively. The absorption rate constant and absolute bioavailability for s.c. zolpidem were 0.083/min and of 84.1%, respectively. The pharmacodynamic parameters for the reinforcement rate, an index of timing performance, were determined by integration of behavioral and pharmacokinetic profiles in a between-subject design using the effect-linked inhibitory sigmoidal E(max) model. The pharmacokinetic-pharmacodynamic analysis revealed that the potency of zolpidem (concentration required to produce 50% maximal effects, IC(50)) in disrupting the timing performance was 0.129 microg/ml. The pharmacodynamic estimates of zolpidem were compared to our previous results for benzodiazepines.

We investigated the pharmacokinetic determinants of the frequency of intravenous cocaine self-administration in 2.5-h sessions. Two groups of rats were implanted with dual catheters that permitted cocaine infusion and blood sampling via the femoral and jugular vein catheters, respectively. Half of the animals in each group self-administered one of the two cocaine unit doses (0.5 and 1 mg/kg/infusion) by pressing a lever under a continuous schedule of reinforcement. To monitor serum cocaine concentrations, the remaining animals received concurrent, response-independent infusions whenever the matched animals self-administered cocaine infusions. Multiple concentration-time data in two successive self-administrations were determined to monitor the extent of fluctuation in concentrations by pharmacokinetic modeling. Behavioral analyses revealed the higher unit dose (1 mg/kg) resulted in less frequent cocaine self-administration, and a longer interinfusion interval, whereas the total doses were similar for the two groups (24.5-27.0 mg/kg/2.5 h). Cocaine decayed biexponentially. Both the values of clearance and terminal elimination rate constant for the self-administration paradigm were significantly greater than those after the bolus cocaine dosing series (0.5 and 1 mg/kg, separated by 3 days). The regularity in cocaine self-administration produced relatively stable serum cocaine concentrations that oscillated between maximum (C(max)) and minimum (C(min)) values regardless of dose size and interinfusion interval. Although the C(max) for the 1-mg/kg unit dose (1.47 microg/ml) was significantly higher than that for the 0.5-mg/kg dose (0.82 microg/ml), the C(min) values between the groups approximated each other (0.28, and 0.34 microg/ml, respectively). Hence, the C(min) is the determinant of the initiation of the next drug-taking behavior.

We characterized the pharmacokinetics of cocaine using both arterial and venous serum data after a bolus dose (2 mg/kg) and two constant-rate infusions (12.24 and 24.48 microg/min) for 2 h in rats. A published behavioral effect was used to investigate the effects of arteriovenous serum concentration differences on pharmacodynamic estimates for the 2 mg/kg dose. Significant temporal arteriovenous serum cocaine and benzoylecgonine (the major metabolite) concentration differences existed after cocaine administrations. The AUCs for arterial serum data were greater than the AUCs for venous data, indicating that cocaine was metabolized more extensively in the venous sampling site. Cocaine's behavioral effect could be directly related to serum concentrations with no hysteresis observed between the effects and arterial or venous serum concentrations. The pharmacodynamic estimates derived from arterial serum data approximated those from the venous data due to the most decline of cocaine's effect occurred in the elimination phase during which serum cocaine concentrations were not significantly different between the two sampling sites.

To accurately assess the mechanism of involvement of the active metabolite norcocaine in the effects of oral cocaine, it is essential to determine the rate and extent of the formation of norcocaine. Although this study was designed specifically for this aim, it was also of interest to characterize the metabolite kinetics of benzoylecgonine for comparative purpose. We first characterized the pharmacokinetics of cocaine, norcocaine, and benzoylecgonine by the i.v. route of administration; all three drugs decayed biexponentially. These pharmacokinetic estimates were then used for determination of the formation of norcocaine and benzoylecgonine after i.v. and p.o. (20-40 mg/kg) cocaine administration. Although t(1/2alpha), and t(1/2beta) were similar across the three compounds, the values of volume of distribution in the central compartment and clearance for benzoylecgonine were much smaller than those of cocaine and norcocaine. Norcocaine was not detected following i.v. cocaine; however, serum norcocaine concentrations were as high as those of oral cocaine. Both routes of cocaine administration produced benzoylecgonine. A pharmacokinetic model for the metabolite kinetics was proposed by sequentially adding the models that most adequately described the formation of each metabolite to the model of cocaine. For oral cocaine, the absolute bioavailability was 3.48%, whereas 6.04 and 2.26% of cocaine were converted to benzoylecgonine and norcocaine, respectively, during first-pass absorption regardless of dose. Furthermore, the majority of norcocaine and 92% of benzoylecgonine were formed during the first-pass absorption, leaving 8% of benzoylecgonine produced in systemic circulation. The profile of norcocaine as a metabolite confirmed the involvement of norcocaine in cocaine's behavioral effects.

Rationale: Environmental factors affect serum drug concentration-effect relations. For example, after midazolam administration, longer pre-session delays imposed in experimental chambers produced differential concentration-effect relations compared to those of shorter delays., Objectives: To evaluate the extent to which serum concentrations determine alprazolam's effects on spontaneous activity in the presence and absence of a differential reinforcement of low rate (DRL 45-s) contingency using pharmacokinetic-pharmacodynamic analysis. Serum concentrations reported here were simulated from our published pharmacokinetic parameters for alprazolam., Methods: One group (n=8) was used to investigate alprazolam's effects on spontaneous activity within the DRL contingency by placing an activity platform beneath each operant chamber to monitor concurrently both spontaneous activity (large and small movements) and DRL performance (shorter-response and reinforcement rates) in 3-h sessions; a parallel group (n=7) was used without the operant context. The concentration-effect relation of the reinforcement rate was compared and contrasted with those of spontaneous activity., Results: Alprazolam decreased large and small movements within the DRL contingency, which corresponded to that of reinforcement rates under the DRL 45-s schedule. In contrast, without the DRL contingency, alprazolam's effects on small movements were short-lived (i.e., 30 min) and no effects on large movements were detected. Hence, the predicted concentration-effect relations for the reinforcement rate function described those of spontaneous activity well within the operant context, but not those without the operant context. Furthermore, the latter showed no correlation between serum alprazolam concentration and large movements; a significant, but low negative correlation for small movements was observed., Conclusions: The duration of alprazolam's action was dependent on not only dose size but also the behavioral measure examined. By imposing the DRL contingency, spontaneous activity behaves as an ideal pharmacodynamic measure (i.e., continuous, sensitive, and objective).

Groups of rats were exposed to daily, 3-h, fixed-time 1-min food-pellet delivery sessions, a procedure that produces overdrinking (schedule-induced polydipsia, SIP). Previous research demonstrated that rats drinking a drug or non-drug solution come to prefer that solution to water if the solution had (a) a past association with either a highly acceptable vehicle (e.g. glucose/saccharin), or (b) allowed rats to eschew drinking an unacceptable solution under SIP conditions. The present experiments show that under the solution-avoidance procedure, preference for a concurrent, alternative solution (0.19 mg/ml lidocaine or 0.24 mg/ml cocaine) occurred when a concentrated quinine solution alternative was either abruptly removed or faded. A concentrated cocaine solution, however, was minimally effective in producing a preference for 0.19 mg/ml lidocaine to water when cocaine concentration was faded. Flavor/nutrient-conditioning (conditioned reinforcement) and solution-eschewing (avoidance) procedures may throw light on the kinds of historical situations that determine the genesis of stable preferences for drugs and other substances.

In 3-hr sessions, the authors investigated the onset, peak, and disappearance of the effects of alprazolam on performance under a differential reinforcement of low rate 45-s schedule in rats. Alprazolam was administered chronically as a daily bolus dose (2 mg/kg) via the intravenous route. Alprazolam decreased the reinforcement rate and increased the shorter response (nonreinforced) rate in a dose- and time-related fashion. Tolerance did not develop to the decreases in reinforcement rate; tolerance to increases in shorter response rate was negligible, occurring only at the low-concentration range. Clinically, an optimal dose regimen should be designed to avoid the tolerance development that occurs in the low serum benzodiazepine concentration range.

Rationale: Oral cocaine is more effective than IV cocaine by pharmacokinetic and pharmacodynamic analysis. One explanation is involvement of the active metabolite, norcocaine, in cocaine's effects., Objectives: To evaluate norcocaine's contribution to oral cocaine's effects, norcocaine's effects as a parent compound were determined and compared to those of cocaine using a differential reinforcement of low rate (DRL 45-s) schedule and spontaneous activity (large and small movements) after IV and PO routes of administration., Methods: The effects of cocaine and norcocaine on DRL performance (shorter-response and reinforcement rates) and spontaneous activity were investigated in 3-h sessions. The changes in effects across time (effect-time profiles) and dose-response curves (DRCs) were constructed to evaluate the duration of action and potency (ED50) of both drugs., Results: Under the DRL 45-s schedule, effect-time profiles showed both drugs via the two routes of administration significantly increasing and decreasing shorter-response rates and reinforcement rates, respectively. However, cocaine produced greater effects on shorterresponse rates than norcocaine, while both drugs produced comparable effects on reinforcement rates. For spontaneous activity, although IV cocaine, PO cocaine, and PO norcocaine dose- and time-dependently increased spontaneous activity, cocaine's effects were more profound than those of norcocaine. Effect-time profiles revealed that the duration of drug action was a function of dose, route, and behavioral paradigm used. According to ED50 values, IV cocaine was more effective than PO cocaine; however, PO cocaine was more effective than IV cocaine as judged by ED50 values corrected for absolute oral bioavailability., Conclusions: Norcocaine's contribution to oral cocaine's effects on DRL performance is evident. Other mechanism(s), such as a greater acute tolerance to IV cocaine's effects than to PO cocaine's effects, can be excluded.

Despite wide use of cumulative-dosing procedures to evaluate dose-response relations, limited attention has been paid to investigating drug concentration-effect relations. We first characterized the pharmacokinetic (PK) parameters for i.v. (2 mg/kg) and oral cocaine (20 and 40 mg/kg) in rats. Cocaine's concentration-time profile for the escalating cumulative-dose regimen was simulated from PK parameters, dose size (1, 2, 7, 20, and 45 mg/kg by the oral route), and dosing interval (tau, 35 min) as well as validated from blood sampling at various time points. This concentration-time profile was integrated with pharmacodynamic (PD) profiles of differential reinforcement of low rate performance and spontaneous activity (large and small movements) under a differential reinforcement of low rate 45-s schedule. Effects on three behavioral measures were characterized by integrated PK-PD models using the sigmoid E(max) (for increases in shorter response rate or large movements) and inhibitory E(max) (for decreases in density of reinforcement) models. But for the intrinsic differences in baseline and efficacy values among the behavioral endpoints, one set of PD parameters (i.e., potency and Hill factors) predicted concentration-effect relations for the three behavioral indices across all five doses. Concurrent monitoring of operant and spontaneous activity behavior within an operant context provides a novel behavioral paradigm to investigate drug effects on spontaneous activity under conditions where a behavioral contingency exists. Additionally, a cumulative-dosing procedure is efficient for determining the entire dose-response relation and provides an ideal mode to study phenomena such as sensitization or tolerance by varying dose size and/or tau.

A sensitive, selective and simple HPLC method with fluorimetric detection is described for quantitating cocaine and its three metabolites in rat serum microsamples (50 microl). Chromatographic separation is achieved on a Hypersil BDS C18 column (100X2.1 mm, 5 microm) with an isocratic mobile phase consisting of methanol-acetonitrile-25.8 mM sodium acetate buffer, pH 2.6, containing 1.0 x 10(-4) M tetrabutylammonium phosphate (14:10:76, v/v/v). The detection limit (0.5 ng/ml) for all the compounds, using direct fluorometric detection operated at excitation and emission wavelengths of 230 and 315 nm, respectively, was approximately five-times lower than that of using a UV detector operated at 235 nm. The effects of ratio of 2-propanol to chloroform in extraction solvents on the recovery and precision for cocaine and its metabolites were systematically examined. The method was used to study the pharmacokinetics of cocaine after administration of intravenous 2 mg/kg and oral 20 mg/kg doses.

We examined the pharmacokinetics and pharmacodynamics of intravenous (1-5 mg/kg) and oral clozapine (2.5-10 mg/kg) in rats (terminal half-life=81.8 min; oral bioavailability=5.32%). Both dose- and concentration-effect relations of clozapine were characterized. Clozapine's effects were similar to those of benzodiazepines because of the similarity in effect-time profiles between the two classes of drugs. The IC(50) value increased as a function of dose; consequently, clozapine's relative potency decreased linearly with the logarithm of AUC((0-infinity)), or bioavailable dose regardless of route of administration. The IC(50) is an index for the sensitivity of behavioral performance to clozapine; relative potency provides an index for estimating the extent of acute tolerance. As IC(50) increases, relative potency decreases, and consequently, acute tolerance increases. Our results demonstrated that greater acute tolerance was observed for i.v. clozapine than for p.o. clozapine; however, clozapine exhibited a single concentration-effect relation across dose and route of administration after correcting for relative potencies.

The effects of post-injection delay time and environmental context on behaviour after subcutaneous administration of 3 mg/kg midazolam were investigated under a differential reinforcement of low rate schedule (i.e. DRL 45 s) in 3 h sessions. Post-injection delays were varied (0-120 min) for two groups of rats placed in either the experimental chamber (group 1) or home cage (group 2) during the pre-session delay times. Midazolam increased shorter-response (inter-response times < 45 s) rates and decreased reinforcement rates in a time-related manner. Reinforcement rate-time profiles were also integrated with parallel pharmacokinetics. Post-injection delays in either environment yielded performances that mirrored the pharmacokinetic profile operative at the corresponding time-delay points. At higher concentrations (> 0.12 microg/ml) the pharmacokinetics of midazolam largely determined the behavioural effects in both groups, regardless of post-injection delays. However, at lower drug concentrations, longer post-injection delays (> 60 min) in the experimental chamber produced both sensitization and tolerance, as measured by greater increases in shorter-response rates and a more rapid return of the reinforcement rate, respectively. Interaction of the discriminative stimulus effects of midazolam with the context probably alters the magnitude of behavioural effects when the delay occurs in the experimental chambers, whereas no such interaction is present in group 2. The DRL schedule with post-injection delays in experimental chambers provides a useful behavioural paradigm for studying both sensitization and tolerance.

Rats were exposed to daily, 3-h, fixed-time 1-min food-pellet delivery sessions, which is a procedure that produces overdrinking (schedule-induced polydipsia). In previous polydipsia studies, rats came to prefer solutions of drug or non-drug agents to concurrently presented water if the agents had first been offered in a glucose-saccharin vehicle that was slowly eliminated (faded), leaving a choice between a substance in water vehicle vs water. In the first experiment, a more rapid vehicle-fading procedure was used to produce a preference for 0.19mg/ml lidocaine to water. In the second experiment, the vehicle was abruptly changed to water, which also resulted in a strong preference for lidocaine solution, although the lidocaine solution volumes ingested for the final sessions were significantly less than in the first experiment. The results are consistent with a conditioned flavor/nutrient preference interpretation for the institution of the lidocaine preference in both experiments. Although flavor/nutrient conditioning can be a sufficient condition for generating a substance preference, a previous experiment showed that it was not a necessary condition.

A single-solvent extraction step high-performance liquid chromatographic method is described for quantitating zolpidem in rat serum microsamples (50 microl). The separation used a 2.1 mm I.D. reversed-phase OD-5-100 C18 column, 5 microm particle size with an isocratic mobile phase consisting of methanol-acetonitrile-26 mM sodium acetate buffer (adjusted to pH 2.0 with 40% phosphoric acid) containing 0.26 mM tetrabutylammonium phosphate (13:10:77, v/v/v). The detection limit was 3 ng/ml for zolpidem using an ultraviolet detector operated at 240 nm. The recovery was greater than 87% with analysis performed in 12 min. The method is simple, rapid, and applicable to pharmacokinetic studies of zolpidem after administering two intravenous bolus doses (1 and 4 mg/kg) in rats.

Dose-response, effect-time and concentration-effect relations of intravenous cocaine (1-4 mg/kg) were investigated on contingency-controlled [fixed-ratio (FR) 70 performance] and unconditioned (locomotor activity) behaviors. Cocaine dose-response curves exhibited decreasing rates of response under the FR 70 schedule but increasing locomotor activity in a dose-related fashion. Effect-time profiles confirmed that these changes were time-dependent and provided additional clarity by mirroring the biexponential decay of cocaine concentrations with time. The duration of action of cocaine was comparatively shorter on locomotor activity than on FR performance. We integrated effect-time profiles of the two behaviors with concentration-time profiles simulated from our previously published pharmacokinetic parameters to derive cocaine's pharmacodynamic parameters. Classical inhibitory Emax and sigmoidal Emax models were used to describe cocaine's effects on FR performance and locomotor activity, respectively. Simultaneous pharmacokinetic-pharmacodynamic modeling reveals evidence of acute tolerance to cocaine in locomotor activity, as indicated by decreasing potency with dose, but not in contingency-controlled behavior.

The pharmacokinetics of alprazolam (ALP) after i.v. and p.o. administration in rats were characterized. ALP decayed biexponentially after the i.v. dose (1.25 mg/kg), but the concentration-time profiles after the p.o. doses (7 and 12.5 mg/kg) exhibited a double-peak phenomenon. The presence of two peaks was confirmed by statistical analysis of the serum concentration data of ALP, as well as by observed double peaks in the serum concentration-time profiles of the two active metabolites (alpha-hydroxyalprazolam and 4-hydroxyalprazolam). An absorption model incorporating a delay site is proposed to describe the data, and the absolute oral bioavailability is estimated to be about 30%. The two peaks were approximately 80 to 115 min apart, and there was a delay in the absorption of close to 80% of oral ALP, regardless of dose. We hypothesize that the mechanism underlying the double-peak phenomenon is due to reduction in gastric motility caused by the muscle relaxant effect of ALP. This hypothesis is supported by the observed longer delay in the appearance of the second peak at the higher p.o. dose. Enterohepatic recycling is precluded from being the underlying mechanism, because of the presence of double peaks after the p.o. doses but not after the i.v. dose. This is the first reported case of double peaks for oral ALP, and this phenomenon has not been reported for other benzodiazepines. The double-peak phenomenon caused by the hypothesized mechanism may have important therapeutic and drug interaction implications, especially because benzodiazepines are commonly coadministered with other drugs.