Time-dependent Concentration Changes Research Articles

Effect of Holder Pasteurization, Mode of Delivery, and Infant's Gender on Fatty Acid Composition of Donor Breast Milk.

Breast milk (BM) plays a crucial role in providing essential fatty acids (FA) and energy for the growing infant. When the mother's own BM is not available, nutritional recommendations suggest donor milk (DM) in clinical and home practices. BM was collected from a variety of donor mothers in different lactation stages. Holder pasteurization (HoP) eliminates potential contaminants to ensure safety. FA content of BM samples from the Breast Milk Collection Center of Pécs, Hungary, were analyzed before and after HoP. HoP decreases the level of C6:0, C8:0, C14:1n-5c, C18:1n-9c, C18:3n-6c, C18:3n-3c, and C20:4n-6c in BM, while C14:0, C16:0, C18:1n-9t, C22:0, C22:1n-9c, C24:0, C24:1n-9c, and C22:6n-3c were found in elevated concentration after HoP. We did not detect time-dependent concentration changes in FAs in the first year of lactation. BM produced for girl infants contains higher C20:2n-6c levels. In the BM of mothers who delivered via cesarean section, C12:0, C15:0, C16:0, C17:0, C18:0, C18:1n-9t, C22:1n-9c levels were higher, while C18:2n-6c, C22:0, C24:0, and C22:6n-3c concentrations were lower compared to mothers who gave birth spontaneously. FAs in BM are constant during the first year of lactation. Although HoP modifies the concentration of different FAs, pasteurized DM provides essential FAs to the developing infant. Current data providing information about the FA profile of BM gives origination to supplementation guidelines.

Numerical modeling and in situ small angle X-ray scattering characterization of ultra-small SPION magnetophoresis in a high field and gradient separator.

Magnetic nanoparticles (MNPs) have recently gained significant attention in various fields, including chemical and biomedical applications, due to their exceptional properties. However, separating MNPs from solution via magnetophoresis is challenging when MNPs are smaller than 50 nm as Brownian forces become on the order of the magnetic forces. In this study, we successfully separated small MNPs (5-30 nm) by utilizing high magnetic fields and gradients generated by economical permanent magnets. In situ small angle X-ray scattering (SAXS) was used to investigate the time-dependent concentration changes in the ferrofluid, and the results validated that only the 30 nm particles experienced particle aggregation or agglomeration, indicating that dipole-dipole interactions did not play a discernable role in the separation process for particles smaller than ∼15 nm. However, numerical simulations have provided further validation that in the absence of particle-particle interactions, even MNPs with diameters less than 15 nm exhibited magnetophoresis that effectively counteracted the effects of Brownian motion.

Vancomycin Elution Kinetics of Four Antibiotic Carriers Used in Orthopaedic Surgery: In Vitro Study over 42 Days.

This study aimed to analyse and compare the vancomycin elution kinetics of four biodegradable, osteoconductive antibiotic carriers used in clinical practice within a 42-day in vitro setting. Carriers A and D already contained vancomycin (1.1 g and 0.247 g), whereas carriers B and C were mixed with vancomycin according to the manufacturer's recommendations (B: 0.83 g and C: 0.305 g). At nine time points, 50% (4.5 mL) of the elution sample was removed and substituted with the same amount of PBS. Probes were analysed with a kinetic microparticle immunoassay. Time-dependent changes in vancomycin concentrations for each carrier and differences between carriers were analysed. Mean initial antibiotic levels were highest for carrier A (37.5 mg/mL) and lowest for carrier B (5.4 mg/mL). We observed time-dependent, strongly negative linear elution kinetics for carriers A (-0.835; p < 0.001), C (-0.793; p < 0.001), and D (-0.853; p < 0.001). Vancomycin concentrations increased from 48 h to 7 d and dropped thereafter in carriers C and D whilst constantly decreasing at any time point for carrier A. Carrier B showed a shallower decrease. Mean antibiotics levels at 42 d were 1.5 mg/mL, 2.6 mg/mL, 0.1 mg/mL, and 0.1 mg/mL for carriers A, B, C, and D. Differences in mean initial and final vancomycin concentrations for carrier A were significantly larger in comparison to C (p = 0.040). A carrier consisting of allogenic bone chips showed the highest vancomycin-to-carrier ratio and the largest elution over the study period. Whilst vancomycin concentrations were still measurable at 42 days for all carriers, carrier A provided a higher drug-to-carrier ratio and a more consistent antibiotic-releasing profile.

#4152 CROSS-OMICS PROFILING OF PROTEOME, METABOLOME, AND TRANSCRIPTOME REVEALS MOLECULE AND CLEARANCE DYNAMICS AND ORIGIN IN PERITONEAL DIALYSIS

Abstract Background and Aims Peritoneal dialysis (PD) effluent is not only a rich source of markers for therapy monitoring and investigation of deregulated processes during PD it is also surprisingly underexplored and therefore poorly defined. Modern high performance mass spectrometry (MS) and sequencing methods allow monitoring of hundreds of analytes in parallel. For understanding PD transport dynamic and pathomechanisms and on a systems biology level, a multi-level omics approach is particularly attractive. Method Samples were obtained from stable patients chronically treated with PD at different time-points of standard 4h peritoneal equilibration tests (PETs). Effluent was collected after the pre-PET (overnight) dwell and at 0h, 1h and 4h dwells. Plasma samples were taken at the 2h PET time point. Effluent was separated into a cellular and cell-free component. Soluble proteins and metabolites in the cell-free compartment were processed using material-specific protocols and standardized LC-MS workflows. The cellular material was subjected to RNA sequencing. The Plasma-Proteome database was used for referencing plasma proteins and estimating plasma concentration. A bioinformatic workflow conjoined information from the datasets to reveal novel insights into the “PD-effluentome”, especially unravelling the origin of proteins and metabolites in PD effluent. Results Metabolomics enabled detecting of 207 unique metabolites in cell-free PD effluent. A mixed-effect ANOVA of all metabolites demonstrated dwell time-dependent concentration changes in 173 metabolites. Post-hoc testing revealed most metabolites to be changed between 1h and overnight time points, followed by 114 and 46 differently concentrated metabolites between 4h and overnight and 1h and 4h, respectively. We quantified 9,797 transcripts in PD-effluent cells and 2,729 proteins in PD effluent. 342 proteins were filtered from plasma, while 800 proteins were attributable to local origin or production. A quantitative analysis of the interaction proteome and cellular transcripts of ∼1700 protein-transcript pairs showed clusters of proteins explained by over-expression in peritoneal cells compared to plasma concentrations. Conclusion Cross-omic profiling of PD effluent can be a valuable approach for revealing small molecule related changes during PD. The exploitation of PD effluent on multiple levels could improve the understanding of pathophysiological molecular processes and transport dynamics in the peritoneal cavity and their role in development of PD complications.

Comprehensive stability study of benzimidazole drug residues in standard solution, muscle and milk

The performance criteria of analytical methods and the necessity for stability analysis to provide the accuracy of the results of the analyzed samples are explained in European Commission Decision 2021/808/EC and the guidance document SANTE/2021/11312. Detection of time-dependent changes in drug concentrations during storage or transport and re-analysis of samples are crucial to obtain high-quality results and reliable data. In this way, it allows toxicologists to interpret the analytical results accurately in drug analyses. The aim of this study was comprehensively to investigate the stability of benzimidazoles (levamisole hydrochloride, albendazole, albendazole-sulfone, albendazole-2-amino sulfone, albendazole sulfoxide, oxfendazole, 5-hydroxythiabendazole, triclabendazole, ketotriclabendazole, thiabendazole, flubendazole, fenbendazole sulfone) in working solutions, muscle and milk samples. For this purpose, long-term stability was evaluated over 6 months and under four different storage conditions (4 °C, −20 °C, 20 °C light and 20 °C dark) in the matrix. The influences of three freeze-thaw cycles, autosampler stability, and 60 min storage at 40 °C were investigated for short-term stability. Simultaneously, the stability of the working solutions were established over 6 months and under five different conditions (4 °C, −20 °C, −80 °C, 20 °C light, and 20 °C dark). It was found that working solutions can be stored at −80 °C or −20 °C, and it is appropriate to prepare the standard working solution freshly once a month. Storage of milk at 4 °C is suitable for some analytes (ABZ-SO, FBZ-SO2, FLUBZ, ABZ, ABZ-NH2-SO2) whereas for the muscle almost all substances were stable only at −20 °C. Some freeze-thaw and short-term stability changes were detected.

Hybridization kinetics of out-of-equilibrium mixtures of short RNA oligonucleotides.

Hybridization and strand displacement kinetics determine the evolution of the base paired configurations of mixtures of oligonucleotides over time. Although much attention has been focused on the thermodynamics of DNA and RNA base pairing in the scientific literature, much less work has been done on the time dependence of interactions involving multiple strands, especially in RNA. Here we provide a study of oligoribonucleotide interaction kinetics and show that it is possible to calculate the association, dissociation and strand displacement rates displayed by short oligonucleotides (5nt–12nt) that exhibit no expected secondary structure as simple functions of oligonucleotide length, CG content, ΔG of hybridization and ΔG of toehold binding. We then show that the resultant calculated kinetic parameters are consistent with the experimentally observed time dependent changes in concentrations of the different species present in mixtures of multiple competing RNA strands. We show that by changing the mixture composition, it is possible to create and tune kinetic traps that extend by orders of magnitude the typical sub-second hybridization timescale of two complementary oligonucleotides. We suggest that the slow equilibration of complex oligonucleotide mixtures may have facilitated the nonenzymatic replication of RNA during the origin of life.

Exercise-induced cardiac troponin release in middle-aged and older athletes with different degrees of coronary atherosclerosis

Abstract Funding Acknowledgements Type of funding sources: Public hospital(s). Main funding source(s): Radboud University Medical CenterAcademic Alliance Fund Maastricht - Nijmegen Background Current sports medical examinations do not appropriately detect occult coronary atherosclerosis in athletes, whereas coronary atherosclerosis is more prevalent in middle-aged and older athletes compared to physically active controls. Therefore, new diagnostic approaches need to be developed to identify individuals at risk. Cardiac troponin (cTn) is the standard biomarker to assess myocardial injury. Exercise is known to increase cTn concentrations and a delayed recovery of cTn concentrations after exercise cessation is suggested to be indicative of occult obstructive coronary artery disease. We hypothesized that the magnitude of exercise-induced cTn elevations may be associated with the degree of coronary atherosclerosis. Purpose To compare serial measures of exercise-induced cTnT elevations across middle-aged and older athletes with different degrees of coronary atherosclerosis. Methods A subgroup of 59 men from the Measuring Athlete’s Risk of Coronary events-2 study (MARC-2, n=291) were invited for this add-on study. All participants had undergone a cardiac computed tomography (CT) scan and coronary CT angiography to determine the prevalence and magnitude of coronary atherosclerosis. Athletes were stratified based on the degree of coronary atherosclerosis: I) n=20 with a coronary artery calcium score (CACS) of 0, II) n=20 with CACS ≥300 or ≥75th MESA-percentile, and III) n=19 with a &amp;gt;50% stenosis in any coronary artery, but without ischemia on a stress imaging tests. Participants performed an individualized exercise test (&amp;gt;1 hour, cycling) until volitional exhaustion. High-sensitivity cTnT concentrations were measured at baseline, after 30 minutes of exercise and at 0, 30, 60, 120, and 180 minutes post-exercise. Results Participants (age 60.8 [57.9-72.6] years, BMI 24.5 [23.9-27.3]) exercised for 76±14 minutes, up to 97.6 [94.4-101.8] % of their expected maximum heart rate. Exercise duration and workload did not differ across groups. Baseline hs-cTnT was detectable in everyone. Exercise induced a 1.98±1.63 fold increase in hs-cTnT concentrations. Peak hs-cTnT concentrations occurred at 180 minutes post-exercise (13.9 [9.4-17.0] ng/L versus 12.1 [10.0-21.5] ng/L versus 13.8 [9.1-19.9] ng/L, for group I, II and III, respectively) and the incidence of hs-cTnT concentrations above the URL (n=9 (45%) vs n=7 (35.0%) vs n=9 (47.4%)) was comparable across groups (p=0.92 and p=0.71, respectively). Patterns of time-dependent changes in hs-cTnT concentrations did not differ across groups (p=.49). Conclusion Exercise-induced elevations in hs-cTnT concentrations did not differ among middle-aged and older athletes with different degrees of coronary atherosclerosis. Our findings suggest that hs-cTnT sampled before, during or within 180 minutes following an exhaustive endurance cycling exercise test is not suitable to detect occult coronary atherosclerosis in athletes.

MO701: Origin of Proteins in Peritoneal Dialysis Explained by a Transcriptomics/Proteomics Cross-Over Analysis

Abstract BACKGROUND AND AIMS Peritoneal dialysis effluent (PDE) is a rich but underexplored source of molecular markers for therapy monitoring and investigation of deregulated processes during PD. Modern high performance mass spectrometry (MS) and sequencing methods allow monitoring of hundreds of analytes in parallel. For understanding PD related processes on a systems biology level, a multilevel omics approach is particularly attractive. Here, we investigate the cellular transcriptome and cell-free proteome of PDE samples in combination with the publicly available Human Plasma Proteome Database to investigate the origin of proteins found in peritoneal dialysis effluent. METHOD Samples were obtained from clinically stable patients on chronic peritoneal dialysis during a highly standardized clinical routine check. The effluent material was separated into a cellular and cell-free component. Soluble proteins in the cell-free compartment were processed using our equalizing and TMT-labeling workflow followed by LC-MS. The cellular material was subjected to RNA sequencing. The Human Plasma Proteome database (peptideatlas.org/hupo/hppp) was used for referencing plasma proteins and estimating plasma concentration. A bioinformatic workflow conjoined information from the datasets to reveal novel insights into the ‘PD effluentome,’ especially clarifying the source of proteins found in PDE. RESULTS Combining two targeted metabolomics methods enabled detecting 207 unique metabolites in cell-free PDE. Metabolites not detected in our samples were included in the panel for in vitro studies of cellular systems. A mixed-effect ANOVA of all metabolites demonstrated dwell time-dependent concentration changes in 173 metabolites. Post-hoc testing revealed most metabolites to be changed between 1 and 16 h [ON] of fluid dwell (160), followed by 114 and 46 differently concentrated metabolites between 4 and 16 h and 1 and 4 h of dwell, respectively. We quantified 9797 transcripts in PD-effluent cells and 2729 solved proteins in PD effluent. A total of 342 proteins were filtered from plasma, while 800 proteins were attributable to local production. A quantitative analysis of the interaction proteome and cellular transcripts of roughly 1700 protein-transcript pairs showed clusters of proteins explained by overexpression in peritoneal cells compared to plasma concentrations. CONCLUSION Multi-omic profiling of PD effluent proved to be a valuable approach for revealing small molecule related changes during PD treatment. The exploitation of PD effluent information on multiple omics levels as identified by our bioinformatic approach has been shown to improve our understanding of the molecular processes in the peritoneal cavity and their role in development of complications for ultimately improving PD therapy. The combinatorial investigation of proteome and transcriptome of PD effluent represents a first step in identifying locally produced proteins for further validation as biomarkers of peritoneal health in peritoneal dialysis patients. Proteins of plasma origin could be tested for their value as diagnostic tools in monitoring treatment success and protein transport over the peritoneal barrier. Our work suggests feasibility of multi-omics approaches to investigate cell-derived biomarkers for their involvement in pathomechanisms relevant in PD.

Lipopolysaccharide challenge following intravenous amino acid infusion in postpartum dairy cows: I. Production, metabolic, and hormonal responses

Postpartum cows experience a nadir in energy and AA deficit early postpartum. At the same time, cows are challenged with inflammatory stimuli and often show heightened immune responsiveness, further increasing their metabolic needs during this critical time. This study investigated the response to a systemic inflammatory stimulus after a 4-d intravenous (IV) AA infusion designed to ameliorate the estimated metabolizable protein (MP) deficit in postpartum cows. Our objectives were to (1) describe the production and metabolic responses to early postpartum IV AA infusion, (2) determine the metabolic and hormonal responses to an acute IV lipopolysaccharide (LPS) challenge in early postpartum cows, and (3) compare these metabolic and hormonal responses between IV AA treated and control cows. Cows (n = 14, 4 ± 1 d in milk) were continuously IV infused for 4 d in a matched-pair randomized controlled design and received IV AA (IVAA) or 0.9% NaCl (CTRL). Treatment with IV AA consisted of 1 g/kg of BW per day of combined essential AA (EAA) and nonessential AA (NEAA). After infusion ended, cows were challenged IV with LPS (0.0625 µg/kg of BW over 1 h), and serial blood samples were collected to quantify AA, metabolite, and hormone concentrations. Amino acid infusion increased plasma EAA and NEAA concentrations and ameliorated the estimated MP deficit but not the metabolizable energy deficit in IVAA cows. Patterns of dry matter intake during infusion were different between groups. Milk yield and milk protein content and yield were unaffected, but IV AA was associated with increased milk fat content and yield of both de novo and preformed fatty acids. Before LPS infusion, plasma EAA and NEAA concentrations were greater in IVAA compared with CTRL. During LPS challenge, plasma AA concentrations decreased to a greater degree in IVAA than CTRL. Glucagon concentrations were greater and glucose concentrations lower in IVAA during challenge; however, previous AA infusion did not affect the time-dependent changes in concentrations of energy metabolites or glucoregulatory hormones. Plasma urea nitrogen concentration increased in both treatments following challenge, although the temporal pattern depended on treatment. Effects of AA infusion on milk fat response were pronounced and likely due to a combination of increased lipolysis and de novo milk fat synthesis. Despite differences in circulating concentrations of nutrients and hormones before challenge, metabolic responses to systemic inflammation did not differ between the 2 treatments. We conclude that AA infusion changed metabolic status and milk fat but did not appear to alter the metabolic response to subsequent systemic inflammation.

Molecular Diffusivity of Click Reaction Components: The Diffusion Enhancement Question.

Micrometer-sized objects are widely known to exhibit chemically driven motility in systems away from equilibrium. Experimental observation of reaction-induced motility or enhancement in diffusivity at the much shorter length scale of small molecules is, however, still a matter of debate. Here, we investigate the molecular diffusivity of reactants, catalyst, and product of a model reaction, the copper-catalyzed azide–alkyne cycloaddition click reaction, and develop new NMR diffusion approaches that allow the probing of reaction-induced diffusion enhancement in nanosized molecular systems with higher accuracy than the state of the art. Following two different approaches that enable the accounting of time-dependent concentration changes during NMR experiments, we closely monitored the diffusion coefficient of reaction components during the reaction. The reaction components showed distinct changes in the diffusivity: while the two reactants underwent a time-dependent decrease in their diffusivity, the diffusion coefficient of the product gradually increased and the catalyst showed only slight diffusion enhancement within the range expected for reaction-induced sample heating. The decrease in diffusion coefficient of the alkyne, one of the two reactants of click reaction, was not reproduced during its copper coordination when the second reactant, azide, was absent. Our results do not support the catalysis-induced diffusion enhancement of the components of the click reaction and, instead, point to the role of a relatively large intermediate species within the reaction cycle with diffusivity lower than that of both the reactants and product molecule.

Postmortem Metabolomics: Strategies to Assess Time-Dependent Postmortem Changes of Diazepam, Nordiazepam, Morphine, Codeine, Mirtazapine and Citalopram

Postmortem redistribution (PMR) can result in artificial drug concentration changes following death and complicate forensic case interpretation. Currently, no accurate methods for PMR prediction exist. Hence, alternative strategies were developed investigating the time-dependent postmortem behavior of diazepam, nordiazepam, morphine, codeine, mirtazapine and citalopram. For 477 authentic postmortem cases, femoral blood samples were collected at two postmortem time-points. All samples were quantified for drugs of abuse (targeted; liquid chromatography-tandem mass spectrometry LC-MS/MS) and characterized for small endogenous molecules (untargeted; gas chromatography-high resolution MS (GC-HRMS). Trends for significant time-dependent concentration decreases (diazepam (n = 137), nordiazepam (n = 126)), increases (mirtazapine (n = 55), citalopram (n = 50)) or minimal median postmortem changes (morphine (n = 122), codeine (n = 92)) could be observed. Robust mathematical mixed effect models were created for the generalized postmortem behavior of diazepam and nordiazepam, which could be used to back-calculate drug concentrations towards a time-point closer to the estimated time of death (caution: inter-individual variability). Significant correlations between time-dependent concentration changes of morphine, mirtazapine and citalopram with individual endogenous molecules could be determined; no correlation was deemed strong enough for successful a posteriori estimation on the occurrence of PMR for specific cases. The current dataset did successfully lead to a significant knowledge gain in further understanding the time-dependent postmortem behavior of the studied drugs (of abuse).

Time-dependent changes in serum concentrations of acyclovir and its metabolite, 9-carboxymethoxymethylguanine, in a patient with suspected acyclovir encephalopathy

Time-dependent changes in serum concentrations of acyclovir and its metabolite, 9-carboxymethoxymethylguanine, in a patient with suspected acyclovir encephalopathy

Time- and Site-Dependent Postmortem Redistribution of Antidepressants and Neuroleptics in Blood and Alternative Matrices.

Postmortem redistribution (PMR) leads to challenges in postmortem case interpretation. Particularly antidepressants and neuroleptics are expected to undergo PMR based on their physico-chemical properties. For the current study, time- and site-dependent PMR of 20 antidepressants and neuroleptics were investigated in humans (authentic cases); five of which are discussed in detail (citalopram, mirtazapine, quetiapine, risperidone and venlafaxine) along with two metabolites (9-OH-risperidone and O-desmethylvenlafaxine). Blood [femoral (pB) and heart blood (HB)] and tissue biopsy samples (lung, kidney, liver, spleen, thigh muscle and adipose tissue) were collected upon admission to the institute utilizing a computed tomography-guided sample collection workflow (t1). Approximately 24h later (t2; mean 23±9.3h), samples from the same body regions were collected manually. Liquid chromatography-tandem mass spectrometry was used for quantification. Most antidepressants and neuroleptics showed significant time-dependent concentration changes indicating the occurrence of PMR. For the first time, two phases of redistribution in pB for quetiapine were proposed (concentration decreases in the early postmortem phase, followed by concentration increases) and contrasting existing literature, both concentration increases and decreases in pB overtime were observed for risperidone and 9-OH-risperidone. Venlafaxine and its metabolite only showed minimal concentration changes, while citalopram exhibited a trend for concentration increases and mirtazapine for concentration decreases in pB overtime. Based on time-dependent tissue data, passive diffusion processes along the muscle-to-pB, liver-to-HB and lung-to-HB concentration gradients could be proposed along with bacterial degradation. Overall, no case interpretation had to be adjusted, which suggests that PMR changes of antidepressants and neuroleptics do not seem to be relevant for forensic case interpretation within the 24h period that was investigated. However, limitations of the current study (e.g., temperature-controlled storage of the bodies) could have led to an underestimation of occurring postmortem changes, hence, interpretation of postmortem results should always be conducted with care, considering PMR phenomena and inter-individual variability.

Postmortem metabolomics: Correlating time-dependent concentration changes of xenobiotic and endogenous compounds.

Postmortem redistribution (PMR) describes the artificial postmortem concentration changes of xenobiotics that may pose major challenges in forensic toxicology. Only a few studies have systematically investigated time-dependent postmortem drug concentration changes so far and the a posteriori estimation of the occurrence of PMR is not yet possible. In this context, the general concept that postmortem biochemical changes in blood might parallel drug redistribution mechanisms seems promising. Thus, the current study investigated the possible correlations between time-dependent postmortem concentration changes of xenobiotic and endogenous compounds; exemplified for authentic morphine (n = 19) and methadone (n = 11) cases. Peripheral blood samples at two time-points postmortem were analyzed for morphine and methadone concentrations and an (un)targeted postmortem metabolomics approach was utilized to combine targeted quantitative analysis of 56 endogenous analytes and untargeted screening for endogenous compounds (characterizing 1174 features); liquid and gas chromatography-mass spectrometry was used respectively. Individual statistically significant correlations between morphine/methadone and endogenous compounds/features could be determined. Hence, the general applicability of the proposed concept could successfully be confirmed. To verify the reproducibility and robustness of the correlating behavior, a larger dataset must be analyzed next. Once a marker/set of markers is found (e.g. robust correlation with specific xenobiotic or xenobiotic class), these could be used as surrogates to further study the time-dependent PMR in a broader variety of cases (e.g. independent of a xenobiotic drug present). A crucial next step will also be the attempt to create a statistical model that allows a posteriori estimation of PMR occurrence of xenobiotics to assist forensic toxicologists in postmortem case interpretation.

Porcine Lung-Derived Extracellular Matrix Hydrogel Properties Are Dependent on Pepsin Digestion Time.

Hydrogels derived from decellularized lungs are promising materials for tissue engineering in the development of clinical therapies and for modeling the lung extracellular matrix (ECM) in vitro. Characterizing and controlling the resulting physical, biochemical, mechanical, and biologic properties of decellularized ECM (dECM) after enzymatic solubilization and gelation are thus of key interest. As the role of enzymatic pepsin digestion in effecting these properties has been understudied, we investigated the digestion time-dependency on key parameters of the resulting ECM hydrogel. Using resolubilized, homogenized decellularized pig lung dECM as a model system, significant time-dependent changes in protein concentration, turbidity, and gelation potential were found to occur between the 4 and 24 h digestion time points, and plateauing with longer digestion times. These results correlated with qualitative scanning electron microscopy images and quantitative analysis of hydrogel interconnectivity and average fiber diameter. Interestingly, the time-dependent changes in the storage modulus tracked with the hydrogel interconnectivity results, while the Young's modulus values were more closely related to average fiber size at each time point. The structural and biochemical alterations correlated with significant changes in metabolic activity of several representative lung cells seeded onto the hydrogels with progressive decreases in cell viability and alterations in morphology observed in cells cultured on hydrogels produced with dECM digested for >12 and up to 72 h of digestion. These studies demonstrate that 12 h pepsin digest of pig lung dECM provides an optimal balance between desirable physical ECM hydrogel properties and effects on lung cell behaviors.

Time-Dependent Changes in THC Concentrations in Deceased Persons

Changes in the concentrations of Δ9-tetrahydrocannabinol (THC) in the postmortem period were investigated in a series of cases by comparing concentrations in blood taken on receipt of the body in the mortuary (admission specimen, AD) with the concentrations obtained in blood taken at autopsy some time later and also from blood specimens taken antemortem. Overall, the median THC concentration in AD blood was 13.7ng/mL (n=239, range LOQ-220), while the median concentration at autopsy was 13.8ng/mL (n=106, range LOQ-810) and 1.9ng/mL (n=147, range LOQ-48) antemortem. Fourteen cases had all three specimens taken from the same decedent. The corresponding AM, AD and PM median concentrations were 4.0 (range LOQ-48), 15.5 (range 4.0-176) and 4.4ng/mL (LOQ-56), respectively. The median elapsed times from AM to AD and AD to PM were 33 and 97.5h, respectively. In contrast, acetaminophen showed no change in blood concentration from AM to AD (6.8 and 6.0mg/L, respectively). These data show large increases in THC concentration in the early postmortem period, followed by a decline, although the median blood concentrations at autopsy were similar to that obtained antemortem. In contrast, when blood was taken from the femoral region, subclavian and heart ventricles sites, in the same case, the THC concentrations, while variable, showed overall no significant difference. These dynamic changes reflect complex phenomenon occurring in deceased persons and will further serve to increase the uncertainty over any interpretation of postmortem THC concentrations.

A comprehensive time course study of tissue nitric oxide metabolites concentrations after oral nitrite administration

Nitrite and nitrate are considered nitric oxide (NO) storage pools. The assessment of their tissue concentrations may improve our understanding of how they attenuate pathophysiological mechanisms promoting disease. We hypothesized that significant differences exist when the tissue concentrations of nitrite, nitrate, and nitrosylated species (RXNO) are compared among different tissues, particularly when nitrite is administered orally because nitrite generates various NO-related species in the stomach. We studied the different time-dependent changes in plasma and tissue concentrations of nitrite, nitrate, and RXNO after oral nitrite 15 mg/kg was administered rats, which were euthanized 15, 30, 60, 120, 240, 480 or 1440 min after nitrite administration. A control group received water. Arterial blood samples were collected and the rats were perfused with a PBS solution containing NEM/DTPA to prevent the destruction of RXNO. After perfusion, heart, aorta, mesenteric artery, brain, stomach, liver and femoral muscle were harvested and immediately stored at −70°C until analyzed for their nitrite, nitrate and RXNO contents using an ozone-based reductive chemiluminescence assay. While nitrite administration did not increase aortic nitrite or nitrate concentrations for at least 60 min, both aorta and mesenteric vessels stored nitrite from 8 to 24 h after its administration and their tissue concentrations increased from 10 to 40-fold those found in plasma. In contrast, the other studied tissues showed only transient increases in the concentrations of these NO metabolites, including RXNO. The differences among tissues may reflect differences in mechanisms regulating cellular influx of nitrite. These findings have important pharmacological and clinical implications.

Stability of ten psychotropic drugs in formalin-fixed porcine liver homogenates

In forensic toxicology studies, drug concentrations must be estimated by the analytical data of formalin-fixed tissues if fresh or frozen tissue specimens are not available. We wished to investigate the stability and time-course of metabolism/degradation of drugs in formalin-fixed tissues using porcine liver homogenates (PLHs) instead of human tissue. Ten psychotropic drugs (amitriptyline, brotizolam, diazepam, diphenhydramine, estazolam, etizolam, levomepromazine, paroxetine, quetiapine and triazolam) were added to PLHs. After the PLHs had been fixed with neutral buffered formalin at room temperature, the concentrations of the drugs in the PLHs were determined by liquid chromatography–tandem mass spectrometry after 3 days, 1 week, 2 weeks, 4 weeks, 2 months, 4 months and 6 months. After 6 months, the residual ratio of amitriptyline, diphenhydramine and quetiapine was 80 %–95 %; that of diazepam, paroxetine and triazolam was 10 %–45 %; and that of brotizolam, etizolam and levomepromazine was 1 %–5 %. Estazolam was not detected from the first day of formalin fixation. These data suggest that the concentrations of drugs in PLHs measured after formalin fixation decreased to varying degrees compared with their initial concentrations. These time-dependent changes in drug concentration were due to degradation during preservation in formalin solution and metabolism by hepatic microsomal enzymes.

Widespread time-dependent changes in tissue cytokine concentrations in brain regions during the acute phase of endotoxemia in mice

Sepsis-associated encephalopathy (SAE) is a diffuse brain dysfunction induced by the systemic response to infection in septic patients. In the present study, we modeled SAE by administering lipopolysaccharide (LPS) intraperitoneally to mice at a concentration of 3.0 mg/kg. We investigated regional preferences for cytokine-mediated brain reactions to endotoxemia and at what time point brain inflammation begins, as well as what cytokines are involved in acute brain reactions. Brains were divided into seven parts: cortex (CTX), olfactory system (Olf), hippocampus (Hip), striatum (Str), diencephalon (Die), brain stem (BS), and cerebellum (CBL). In each brain region, we determined the tissue concentrations of 11 cytokines: CCL2, CCL3, CCL11, CXCL1, CXCL2, CXCL9, CXCL10, G-CSF, IL-1β, IL-6, and TNF-α, in mice injected with LPS or saline, at 1, 4, and 24 h after injection using multiplex cytokine assays. Every brain region responded with the production of multiple cytokines to LPS-induced systemic inflammation during the acute phase (4–24 h) after LPS injection. IL-6, CCL2, CCL3, CXCL1, CXCL2, CXCL9, and TNF-α were “early cytokines” that increased only at 4 h but not at 24 h after LPS injection in most brain regions. CCL11, CXCL10, and G-CSF were “late cytokines” that were elevated up to 24 h after LPS injection in selected brain regions. The regions Olf, Hip, and Die were the most responsive to endotoxemia; these regions produced ten cytokines and continued to produce three “late cytokines” up to 24 h after LPS injection. Str was the least responsive to endotoxemia. The widespread nature of brain cytokine production explains the characteristics of SAE. Further studies on the roles of CCL11, CXCL10, and G-CSF may be especially important in terms of potential prevention of SAE between 4 and 24 h after the onset of sepsis.

A Pilot Study on Continuous Infusion of 4% Albumin in Critically Ill Patients: Impact on Nosocomial Infection via a Reduction Mechanism for Oxidized Substrates.

In a prospective, randomized, open-label trial, shock patients were allocated to receive either continuously 4% albumin or intermittently 20% albumin, as long as they were infused with norepinephrine. A translational study including in vivo and in vitro analyses of albumin-vasostatin-I interactions is reported. A tertiary ICU caring for 1,000 patients per year. Fifty shock patients with serum albumin less than 20 g/L. In vivo colonization and nosocomial infections were recorded and time-dependent changes in serum albumin, chromogranin A, and vasostatin-I concentrations as well. In vitro, we studied biochemical albumin-vasostatin-I relationship using biochemical methods. Over 18 days, we recorded a decrease in colonization (four vs 12 episodes; p = 0.035) and nosocomial infection frequency (two vs 13 episodes; p = 0.002) in patients infused continuously 4% albumin versus controls. In vitro, albumin interacts with the disulfide loop vasostatin-I (residues 17-40) and continuous 4% albumin infusion restores its oxidative status required for antimicrobial activity. Continuous 4% albumin is effective in reducing care-related infections in shock patients by increasing the availability of antimicrobial vasostatin-I. This might guide future care of shock patients.