Enzyme-mediated drug-drug interactions: a review of in vivo and in vitro methodologies, regulatory guidance, and translation to the clinic.

Enzyme-mediated pharmacokinetic drug-drug interactions can be caused by altered activity of drug metabolizing enzymes in the presence of a perpetrator drug, mostly via inhibition or induction. We identified a gap in the literature for a state-of-the art detailed overview assessing this type of DDI risk in the context of drug development. This manuscript discusses in vitro and in vivo methodologies employed during the drug discovery and development process to predict clinical enzyme-mediated DDIs, including the determination of clearance pathways, metabolic enzyme contribution, and the mechanisms and kinetics of enzyme inhibition and induction. We discuss regulatory guidance and highlight the utility of in silico physiologically-based pharmacokinetic modeling, an approach that continues to gain application and traction in support of regulatory filings. Looking to the future, we consider DDI risk assessment for targeted protein degraders, an emerging small molecule modality, which does not have recommended guidelines for DDI evaluation. Our goal in writing this report was to provide early-career researchers with a comprehensive view of the enzyme-mediated pharmacokinetic DDI landscape to aid their drug development efforts.

Assessing and mitigating pH-mediated DDI risks in drug development - formulation approaches and clinical considerations.

pH-mediated drug-drug interactions (DDI) is a prevalent DDI in drug development, especially for weak base compounds with highly pH-dependent solubility. FDA has released a guidance on the evaluation of pH-mediated DDI assessments using in vitro testing and clinical studies. Currently, there is no common practice of ways of testing across the academia and industry. The development of biopredictive method and physiologically-based biopharmaceutics modeling (PBBM) approaches to assess acid-reducing agent (ARA)-DDI have been proven with accurate prediction and could decrease drug development burden, inform clinical design and potentially waive clinical studies. Formulation strategies and careful clinical design could help mitigate the pH-mediated DDI to avoid more clinical studies and label restrictions, ultimately benefiting the patient. In this review paper, a detailed introduction on biorelevant dissolution testing, preclinical and clinical study requirement and PBPK modeling approaches to assess ARA-DDI are described. An improved decision tree for pH-mediated DDI is proposed. Potential mitigations including clinical or formulation strategies are discussed.

Observations and properties of the first laboratory fusion experiment to exceed a target gain of unity.

An indirect-drive inertial fusion experiment on the National Ignition Facility was driven using 2.05 MJ of laser light at a wavelength of 351 nm and produced 3.1±0.16 MJ of total fusion yield, producing a target gain G=1.5±0.1 exceeding unity for the first time in a laboratory experiment [Phys. Rev. E 109, 025204 (2024)10.1103/PhysRevE.109.025204]. Herein we describe the experimental evidence for the increased drive on the capsule using additional laser energy and control over known degradation mechanisms, which are critical to achieving high performance. Improved fuel compression relative to previous megajoule-yield experiments is observed. Novel signatures of the ignition and burn propagation to high yield can now be studied in the laboratory for the first time.

Reducing dietary crude protein: Effects on digestibility, nitrogen balance, and blood metabolites in late-lactation Holstein cows.

Our objectives were to determine the effects of reducing dietary CP concentration on nutrient digestibility, rumen function, N balance, and serum AA concentration for dairy cows in late lactation. At the initiation of the experimental period, we stratified Holstein cows (n = 128; mean ± SD 224 ± 54 DIM) by parity and days pregnant (86 ± 25 d) and assigned them to 1 of 16 pens. For 3 wk, all cows received a covariate diet containing 16.9% CP (DM basis). For the subsequent 12 wk, we assigned pens to 1 of 4 treatments containing 16.2%, 14.4%, 13.4%, or 11.9% CP (DM basis) in a randomized complete block design. Diets were fed as a TMR once daily. To reduce dietary CP, we replaced soybean meal with soybean hulls in the concentrate mix (DM basis). Diet evaluations suggested that several EAA, especially His, limited productivity as dietary CP declined. Digestibility of DM and CP decreased linearly with dietary CP reduction. Digestibility of NDF and potentially digestible NDF tended to respond in a quadratic pattern with the greatest digestibility at intermediate treatments. The reduction in dietary CP did not affect ruminal pH, but ruminal ammonia-N and branched-chain VFA concentrations declined linearly. The concentration of milk urea-N and plasma urea-N, secretion of milk N, and excretions of fecal N, urinary N, urinary urea-N, and unaccounted N decreased linearly with the reduction in dietary CP concentration. Urinary N expressed as a percentage of N intake was unaffected by dietary CP. Serum concentrations of total essential AA and NEAA were unaffected by dietary CP concentration. However, the ratio of essential to NEAA decreased with decreasing dietary CP. Serum 3-methylhistidine concentration increased linearly with decreasing dietary CP concentration, indicating greater skeletal muscle breakdown. Although our trial confirmed that reducing dietary CP decreased absolute excretion of urinary N, diet evaluations suggested that milk protein production decreased as certain essential AA became increasingly limited. Thus, reduced-CP diets have the potential to lessen reactive-N outputs of late-lactation cows, but more research is needed to design diets that minimize deleterious effects on productivity.

Crude protein oscillation in diets adequate and deficient in metabolizable protein: Effects on nutrient digestibility, nitrogen balance, plasma amino acids, and greenhouse gas emissions.

Reducing dietary CP is a well-established means to improve N use efficiency. Yet, few studies have considered if transient restrictions in dietary CP could reduce the environmental footprint of late-lactation cows. We hypothesized that the effects of CP feeding pattern on digestibility and environmental outputs would be amplified at lower dietary CP. We tested CP levels below and near predicted requirements (low protein [LP], 13.8%; high protein [HP], 15.5%) offered in 2 feeding patterns: where diets alternated ±1.8 percentage units CP every 2 d (oscillating [OF]) or remained static. Our study used a 2 × 2 factorial design with 16 mid- to late-lactation Holsteins (mean = 128, SD = 12 DIM), divided into rumen-cannulated (n = 8) and noncannulated subsets (n = 8). For each 28-d experimental period, we recorded feed intake and milk production and took samples of orts (1×/d) and milk (2×/d) for 4 d. For the cannulated subset, we measured and sampled from the total mass of feces and urine production and collected plasma 2×/d across 4 d. For the noncannulated subset, we sampled carbon dioxide and methane emissions 3×/d for 4 d. For each subset, we fit linear mixed models with fixed effects for CP level, CP feeding pattern, the interaction of CP level and CP feeding pattern, period, and a random effect for cow. For plasma and urinary urea-N, we conducted time series analysis. Contrary to our hypothesis, we found no evidence that dietary CP level and CP feeding pattern interacted to influence N balance, nutrient digestibility, or gas emissions. Results showed HP resulted in similar milk N but increased manure N, reducing N use efficiency (milk true protein N/intake N) relative to LP. For OF, urea-N in urine and plasma peaked 46 to 52 h after the first higher-CP phase feeding. Nutrient digestibility and gas emissions were similar across treatments, except CO2 production was greater for OF-HP. In summary, measured variables were minimally affected by dietary CP alternating ±1.8 percentage units every 48 h, even when average dietary CP was fed below predicted requirements (LP). Although our findings suggest that mid- to late-lactation cows are resilient to oscillation in dietary CP, oscillating CP neither reduced the environmental footprint by improving nutrient use efficiencies nor reduced the potential for direct and indirect greenhouse gas emissions.

Dynamic lactation responses to dietary crude protein oscillation in diets adequate and deficient in metabolizable protein in Holstein cows.

Limited research has examined the interaction between dietary crude protein (CP) level and CP feeding pattern. We tested CP level (low protein [LP], 13.8%; high protein [HP], 15.5% CP, dry matter [DM] basis) and CP feeding pattern (OF = oscillating, SF = static) using a 2 × 2 factorial in 16 mid- to late-lactation Holsteins (initially 128 ± 12 d in milk; mean ± SD). Cows ate total mixed rations formulated by exchanging soy hulls and ground corn with solvent soybean meal to keep constant ratios of neutral detergent fiber to starch (1.18:1), rumen-degradable protein to CP (0.61:1), and forage-to-concentrate (1.5:1) in DM. The OF treatments alternated diets every 48 h to vary CP above and below the mean CP level (OF-LP = 13.8% ± 1.8%; OF-HP = 15.5% ± 1.8% CP [DM basis]) whereas diets were constant in SF (SF-LP = 13.8%; SF-HP = 15.5% CP [DM basis]). In four 28-d periods, 8 rumen-cannulated and 8 noncannulated cows formed 2 Latin rectangles. On d 25 to 28 of each period, each cow's feed intake and milk production were recorded, and samples were taken of orts (1×/d) and milk (2×/d). We fit linear mixed models with fixed CP level, CP feeding pattern, and period effects, and a random intercept for cow, computing least squares means and standard errors. Neither CP level, CP feeding pattern, nor the interaction affected DM intake, feed efficiency, or production of milk, fat- and protein-corrected milk (FPCM), fat, true protein, or lactose. Milk urea-N (MUN) yield was lesser for LP. The LP and OF conditions decreased MUN concentration. The CP level tended to interact with CP feeding pattern so that milk protein concentration was greatest for OF-HP. The OF and LP conditions increased the ratio of true protein to MUN yield. Within OF, cosinor mixed models of selected variables showed that cows maintained production of FPCM across dietary changes, but MUN followed a wave-pattern at a 2-d delay relative to dietary changes. A tendency for lesser MUN with OF contradicted prior research and suggested potential differences in urea-N metabolism between OF and SF. Results showed that cows maintained production of economically-relevant components regardless of CP feeding pattern and CP level. Contrary to our hypothesis, the effects of 48-h oscillating CP were mostly consistent across CP levels, suggesting that productivity is resilient to patterned variation in dietary CP over time even when average CP supply is low (13.8% of DM) and despite 48 h restrictions at 12.2% CP.

Epigenetics in drug disposition & drug therapy: symposium report of the 24th North American meeting of the International Society for the Study of Xenobiotics (ISSX).

The 24th North American International Society for the Study of Xenobiotics (ISSX) meeting, held virtually from September 13 to 17, 2021, embraced the theme of "Broadening Our Horizons." This reinforces a key mission of ISSX: striving to share innovative science related to drug discovery and development. Session speakers and the ISSX New Investigators Group, which supports the scientific and professional development of student and early career ISSX members, elected to highlight the scientific content presented during the captivating session titled, "Epigenetics in Drug Disposition & Drug Therapy." The impact genetic variation has on drug response is well established; however, this session underscored the importance of investigating the role of epigenetics in drug disposition and drug discovery. Session speakers, Drs. Ning, McClay, and Lazarus, detailed mechanisms by which epigenetic players including long non-coding RNA (lncRNAs), microRNA (miRNAs), DNA methylation, and histone acetylation can alter the expression of genes involved in pharmacokinetics, pharmacodynamics, and toxicity. Dr. Ning detailed current knowledge about miRNAs and lncRNAs and the mechanisms by which they can affect the expression of drug metabolizing enzymes (DMEs) and nuclear receptors. Dr. Lazarus discussed the potential role of miRNAs on UDP-glucuronosyltransferase (UGT) expression and activity. Dr. McClay provided evidence that aging alters methylation and acetylation of DMEs in the liver, affecting gene expression and activity. These topics, compiled by the symposium organizers, presenters, and the ISSX New Investigators Group, are herein discussed, along with exciting future perspectives for epigenetics in drug disposition and drug discovery research.

Complex Cytochrome P450 Kinetics Due to Multisubstrate Binding and Sequential Metabolism. Part 1. Theoretical Considerations.

Complexities in P450-mediated metabolism kinetics include multisubstrate binding, multiple-product formation, and sequential metabolism. Saturation curves and intrinsic clearances were simulated for single-substrate and multisubstrate models using derived velocity equations and numerical solutions of ordinary differential equations (ODEs). Multisubstrate models focused on sigmoidal kinetics because of their dramatic impact on clearance predictions. These models were combined with multiple-product formation and sequential metabolism, and simulations were performed with random error. Use of single-substrate models to characterize multisubstrate data can result in inaccurate kinetic parameters and poor clearance predictions. Comparing results for use of standard velocity equations with ODEs clearly shows that ODEs are more versatile and provide better parameter estimates. It would be difficult to derive concentration-velocity relationships for complex models, but these relationships can be easily modeled using numerical methods and ODEs. SIGNIFICANCE STATEMENT: The impact of multisubstrate binding, multiple-product formation, and sequential metabolism on the P450 kinetics was investigated. Numerical methods are capable of characterizing complicated P450 kinetics.

Complex Cytochrome P450 Kinetics Due to Multisubstrate Binding and Sequential Metabolism. Part 2. Modeling of Experimental Data.

Three CYP3A4 substrates, midazolam, ticlopidine, and diazepam, display non-Michaelis-Menten kinetics, form multiple primary metabolites, and are sequentially metabolized to secondary metabolites. We generated saturation curves for these compounds and analyzed the resulting datasets using a number of single-substrate and multisubstrate binding models. These models were parameterized using rate equations and numerical solutions of the ordinary differential equations. Multisubstrate binding models provided results superior to single-substrate models, and simultaneous modeling of multiple metabolites provided better results than fitting the individual datasets independently. Although midazolam datasets could be represented using standard two-substrate models, more complex models that include explicit enzyme-product complexes were needed to model the datasets for ticlopidine and diazepam. In vivo clearance predictions improved markedly with the use of in vitro parameters from the complex models versus the Michaelis-Menten equation. The results highlight the need to use sufficiently complex kinetic schemes instead of the Michaelis-Menten equation to generate accurate kinetic parameters. SIGNIFICANCE STATEMENT: The metabolism of midazolam, ticlopidine, and diazepam by CYP3A4 results in multiple metabolites and sequential metabolism. This study evaluates the use of rate equations and numerical methods to characterize the in vitro enzyme kinetics. Use of complex cytochrome P450 kinetic models is necessary to obtain accurate parameter estimates for predicting in vivo disposition.

Enzyme Kinetics, Pharmacokinetics, and Inhibition of Aldehyde Oxidase.

Aldehyde oxidase (AO) has emerged as an important drug metabolizing enzyme over the last decade. Several compounds have failed in the clinic because the clearance or toxicity was underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. Metabolic products from AO-catalyzed oxidation are generally nonreactive and often they have much lower solubility. AO metabolism is not limited to oxidation as AO can also catalyze reduction of oxygen and nitrite. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. AO is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite, and hydroxamic acids. These reductive transformations may cause toxicity due to the formation of reactive metabolites. Moreover, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening.

Aldehyde Oxidase Contributes to All-Trans-Retinoic Acid Biosynthesis in Human Liver.

All-trans-retinoic acid (atRA) is a critical endogenous signaling molecule. atRA is predominantly synthesized from retinaldehyde by aldehyde dehydrogenase 1A1 (ALDH1A1), but aldehyde oxidase (AOX) may also contribute to atRA biosynthesis. The goal of this study was to test the hypothesis that AOX contributes significantly to atRA formation in human liver. Human recombinant AOX formed atRA from retinaldehyde (Km ∼1.5 ± 0.4 µM; kcat ∼3.6 ± 2.0 minute-1). In human liver S9 fractions (HLS9), atRA formation was observed in the absence of NAD+, suggesting AOX contribution to atRA formation. In the presence of NAD+, Eadie-Hofstee plots of atRA formation in HLS9 indicated that two enzymes contributed to atRA formation. The two enzymes were identified as AOX and ALDH1A1 based on inhibition of atRA formation by AOX inhibitor hydralazine (20%-50% inhibition) and ALDH1A1 inhibitor WIN18,446 (50%-80%inhibition). The expression of AOX in HLS9 was 9.4-24 pmol mg-1 S9 protein, whereas ALDH1A1 expression was 156-285 pmol mg-1 S9 protein measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification of signature peptides. The formation velocity of atRA in the presence of NAD+ correlated significantly with the expression of ALDH1A1 and AOX protein. Taken together, the data show that both AOX and ALDH1A1 contribute to atRA biosynthesis in the human liver, with ALDH1A1 being the high-affinity, low-capacity enzyme and AOX being the low-affinity, high-capacity enzyme. The results suggest that in the case of ALDH1A dysfunction or excess vitamin A, AOX may play an important role in regulating hepatic vitamin A homeostasis and that inhibition of AOX may alter atRA biosynthesis and signaling. SIGNIFICANCE STATEMENT: This study provides direct evidence to show that human AOX converts retinaldehyde to atRA and contributes to hepatic atRA biosynthesis. The finding that AOX may be responsible for 20%-50% of overall hepatic atRA formation suggests that alterations in AOX activity via drug-drug interactions, genetic polymorphisms, or disease states may impact hepatic atRA concentrations and signaling and alter vitamin A homeostasis.

Animal sciences undergraduate education since the ASAS centennial: a national survey and scoping review.

The rapid pace of advancement in animal sciences is drastically changing conditions for undergraduate teaching and learning in the discipline. Shortly after the American Society of Animal Science (ASAS) centennial, we conducted a national survey of 90 faculty instructors from 49 academic institutions to assess their perceptions of emerging teaching topics. Participants rated 18 learning outcomes (LO) and 16 types of courses and experiences (CE) with respect to their importance and the adequacy of available offerings. This study presents the results of the survey along with a scoping review of animal sciences teaching and learning publications since 2008 (n = 71). Results indicated that discipline-specific competencies and core experiential learning remain central to animal sciences teaching and identified several distinct needs for research. Namely, we suggest that future research in animal sciences teaching and learning 1) develop animal-science-specific expertise on a greater variety of pedagogies, 2) validate improved methods for assessing transferable skills, 3) expand pedagogical knowledge of emerging topics (e.g., sustainability, data science, welfare science, social science), and 4) deepen and broaden animal sciences' teaching and learning identity through theory-building work and collaborations across instructors, disciplines, and institutions.

Treatment and preliminary outcomes of 150 acute care patients with COVID-19 in a rural health system in the Dakotas.

The majority of available US-published reports present populations with community spread in urban areas. The objective of this report is to describe a rural healthcare system's utilisation of therapeutic options available to treat Coronavirus Disease 2019 (COVID-19) and subsequent patient outcomes. A total of 150 patients were treated for COVID-19 at three hospitals in the Dakotas from 21 March 2020 to 30 April 2020. The most common pharmacological treatment regimens administered were zinc, hydroxychloroquine plus azithromycin and convalescent plasma. Adjunctive treatments included therapeutic anticoagulation, tocilizumab and corticosteroids. As of 1 June 2020, 127 patients have survived to hospital discharge, 12 patients remain hospitalised and 11 patients have expired. The efficacy of hydroxychloroquine and azithromycin use has yet to be determined but was not without risks of corrected QT interval prolongation and arrhythmias in our cohort. We did not appreciate any adverse effects that appeared related to tocilizumab or convalescent plasma administration in those patient subsets. These findings may provide insight into disease severity and treatment options in the rural setting with limited resources to participate in clinical trials and encourage larger comparative studies evaluating treatment efficacy.

Short communication: Effect of glucose infusion dose and stage of lactation on glucose tolerance test kinetics in lactating dairy cows.

The objective for this study was to determine the effect of glucose dose and days following peak milk yield on plasma glucose, serum insulin, and plasma nonesterified fatty acids (NEFA) kinetics during an intravenous glucose tolerance test (IVGTT) in lactating dairy cattle. Six lactating Holstein dairy cows (3 primiparous and 3 multiparous) were assigned to 2 squares and received 0.092, 0.15, or 0.3 g of glucose/kg of body weight (BW) during an IVGTT at 74 and 221 d in milk (DIM), representing early (post-peak) lactation and mid lactation, respectively. Treatments were applied in a replicated Latin square design using contiguous 7-d periods within each stage of lactation. Milk production and dry matter intake were determined daily during the first 6 d of each period. The IVGTT was performed on d 7. For the IVGTT, cows were prepared with indwelling catheters in each jugular vein, and blood samples were collected at -15, -10, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min relative to the glucose infusion. Samples were analyzed for plasma glucose, serum insulin, and plasma NEFA concentrations. Increasing the glucose dose during the IVGTT increased plasma glucose area under the curve (AUC), decreased glucose half-life, and increased maximal plasma glucose concentrations in plasma during the IVGTT. Greater glucose dose during the IVGTT elevated serum insulin AUC and increased nadir NEFA concentrations. Maximal plasma glucose concentration during the IVGTT was lower, whereas maximum NEFA concentration, NEFA AUC, and NEFA clearance rate were greater at 74 than at 221 DIM. Only glucose half-life was responsive to stage of lactation × glucose dose effects during the IVGTT, and the decrease in glucose half-life with increasing glucose dose was greater at 74 than at 221 DIM. Glucose AUC was greater and NEFA AUC lower for cows at 74 than at 221 DIM. For the doses tested, a glucose dose greater than 0.092 g/kg of BW resulted in peak blood glucose concentration that exceeded the previously reported renal glucose excretion threshold of 8.3 mM. There is a need for accompanying data to determine if this is the case for the glucose doses evaluated in this experiment. Based on maximal peak glucose concentrations and effects on glucose half-life, we identify 0.092 g of glucose/kg of BW (0.46 g/kg of metabolic body weight) as the preferred dose for the IVGTT for cows at 74 and 221 DIM in this study.

Rationale and study design for lifestyle intervention in preparation for pregnancy (LIPP): A randomized controlled trial.

INTRODUCTION: Maternal obesity increases neonatal risk for obesity and metabolic syndrome later in life. Prior attempts to break this intergenerational obesity cycle by limiting excessive gestational weight gain have failed to reduce neonatal adiposity. Alternatively, pre-conception lifestyle interventions may improve the in utero metabolic milieu during early pregnancy leading to improved fetal outcomes. This randomized controlled trial (RCT) is evaluating whether a lifestyle intervention to reduce weight and improve maternal metabolism in preparation for pregnancy (LIPP) attenuates neonatal adiposity, compared to standard medical advice. MATERIAL AND METHODS: Overweight/class 1 obese women after a previous pregnancy, ~12 weeks postpartum, preparing for a subsequent pregnancy, will be block randomized (1:1) to either LIPP or standard of care in a parallel design. Randomization is stratified by lactation status and overweight vs. class 1 obesity. The LIPP program consists of intensive short-term weight loss followed by weight maintenance until conception using supervised exercise and a low glycemic Mediterranean diet. PRIMARY OUTCOMES: Group differences in neonatal adiposity at birth assessed by PEA POD and placental mitochondrial lipid metabolism. SECONDARY OUTCOMES: Group differences in maternal pregravid and gestational body composition, insulin sensitivity, ß-cell function, fasting metabolic and inflammatory biomarkers, and overall quality of life. Exploratory outcomes include umbilical cord blood insulin resistance, lipid profile and inflammation. DISCUSSION: This RCT will determine the efficacy of maternal weight loss prior to pregnancy on reducing neonatal adiposity. Findings may change standard obstetrical care by providing Level 1 evidence on lifestyle interventions improving neonatal outcomes for women planning for pregnancy. CLINICAL TRIAL REGISTRATION: NCT03146156.

Drinking water quality in the glacial aquifer system, northern USA.

Groundwater supplies 50% of drinking water worldwide, but compromised water quality from anthropogenic and geogenic contaminants can limit usage of groundwater as a drinking water source. Groundwater quality in the glacial aquifer system, USA (GLAC), is presented in the context of a hydrogeologic framework that divides the study area into 17 hydrogeologic terranes. Results are reported at aquifer-system scale and regional (terrane) scale. This paper presents a quantitative assessment of groundwater quality in the GLAC using data from numerous sources for samples collected 2005-2013, compared to health-based and aesthetic (non-health) benchmarks, and evaluated with areal and population metrics. Concentrations above a benchmark are considered high. Trace elements are widespread across the study area, with an estimated 5.7 million people relying on groundwater with high concentrations of one or more trace elements; manganese and arsenic are most often at high concentration. Nitrate is found at high concentration in 4.0% of the study area, serving about 740 thousand people. Organic compounds including pesticides and volatile organic compounds are high in 2.0% of the assessed study area, with about 870 thousand people relying on groundwater with high concentrations of an organic compound. High arsenic and manganese concentrations occur primarily in the terranes with thick, stratigraphically complex, fine-grained glacial sediment, coincident with groundwater under reducing conditions (indicated by iron concentrations >100 µg/L); high nitrate is uncommon in those same terranes. When nitrate is high in thick, fine-grained, complex terranes, though, it is much more commonly associated with groundwater under more oxidizing conditions. Common geogenic trace elements occur at high concentration due to characteristic geologic and geochemical conditions. Conversely, anthropogenic nitrate and organic compounds are introduced at or near the land surface. High concentrations of nitrate or organic compounds are generally limited to areas in proximity where people live and use the chemicals.

Time Course of Aldehyde Oxidase and Why It Is Nonlinear.

Many promising drug candidates metabolized by aldehyde oxidase (AOX) fail during clinical trial owing to underestimation of their clearance. AOX is species-specific, which makes traditional allometric studies a poor choice for estimating human clearance. Other studies have suggested using half-life calculated by measuring substrate depletion to measure clearance. In this study, we proposed using numerical fitting to enzymatic pathways other than Michaelis-Menten (MM) to avoid missing the initial high turnover rate of product formation. Here, product formation over a 240-minute time course of six AOX substrates-O6-benzylguanine, N-(2-dimethylamino)ethyl)acridine-4-carboxamide, zaleplon, phthalazine, BIBX1382 [N8-(3-Chloro-4-fluorophenyl)-N2-(1-methyl-4-piperidinyl)-pyrimido[5,4-d]pyrimidine-2,8-diamine dihydrochloride], and zoniporide-have been provided to illustrate enzyme deactivation over time to help better understand why MM kinetics sometimes leads to underestimation of rate constants. Based on the data provided in this article, the total velocity for substrates becomes slower than the initial velocity by 3.1-, 6.5-, 2.9-, 32.2-, 2.7-, and 0.2-fold, respectively, in human expressed purified enzyme, whereas the K m remains constant. Also, our studies on the role of reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, show that ROS did not significantly alter the change in enzyme activity over time. Providing a new electron acceptor, 5-nitroquinoline, did, however, alter the change in rate over time for mumerous compounds. The data also illustrate the difficulties in using substrate disappearance to estimate intrinsic clearance.

Kinetic mechanism of time-dependent inhibition of CYP2D6 by 3,4-methylenedioxymethamphetamine (MDMA): Functional heterogeneity of the enzyme and the reversibility of its inactivation.

We investigate the mechanism of time-dependent inhibition (TDI) of human cytochrome P450 2D6 (CYP2D6) by 3,4-methylenedioxymethamphetamine (MDMA, ecstasy), one of the most widespread recreational drugs of abuse. In an effort to unravel the kinetic mechanism of the formation of metabolic inhibitory complex (MIC) of CYP2D6 with MDMA-derived carbene we carried out a series of spectrophotometric studies paralleled with registration of the kinetics of time-dependent inhibition (TDI) in CYP2D6-incorporated proteoliposomes. The high amplitude of spectral signal in this system allowed us to characterize the spectral properties of the formed MIC in details and obtain an accurate spectral signature of MIC formation. This information was then used in the studies with CYP2D6-containing microsomes of insect cells (CYP2D6 Supersomes™). Our results demonstrate that in both systems the formation of the ferrous carbene-derived MIC is relatively slow, reversible and is not associated with the accumulation of the ferric carbene intermediate, as takes place in the case of CYP3A4 and podophylotoxin. Furthermore, the limited amplitude of MIC formation suggests that only a fraction (∼50%) of spectrally detectable CYP2D6 in both proteoliposomes and Supersomes participates in the formation of MIC and is therefore involved in the MDMA metabolism. This observation reveals yet another example of a cytochrome P450 that exhibits persistent functional heterogeneity of its population in microsomal membranes. Our study provides a solid methodological background for further mechanistic studies of MIC formation in human liver microsomes and demonstrates that the potency and physiological relevance of MDMA-dependent TDI of CYP2D6 may be overestimated.

Short communication: Decreasing the dietary ratio of n-6 to n-3 fatty acids increases the n-3 concentration of peripheral blood mononuclear cells in weaned Holstein heifer calves.

Utilization of nutrients to improve overall heifer health is of interest because of the importance of replacement heifers to the dairy industry. The objective of our study was to compare the effect of supplementation of dietary n-3 and n-6 fatty acids (FA) on FA concentrations in peripheral blood mononuclear cells (PBMC) of Holstein calves. Twenty-seven Holstein heifer calves (107 ± 2.6 d of age; 142.6 ± 6.5 kg of body weight) from the university research and teaching herd were randomly assigned to a common TMR supplemented with 1 of 3 treatments: Ca salts of flaxseed FA (Virtus Nutrition, Corcoran, CA) containing 35% 18:3 n-3 (N3), Ca salts of soybean FA (Virtus Nutrition) containing 50% 18:2 n-6 (N6), or a 50:50 mix of N3 and N6. Treatments were supplemented with FA at 4% of dietary dry matter and fed for 30 d. Feed intake was recorded daily, and body weight, wither height, and body condition score were measured weekly throughout the study. On d 28 heifers were vaccinated with a Pasteurella vaccine and the temperature response to the vaccine was recorded. Blood was collected on d 0 and 28 for PBMC isolation. After total lipid extraction and FA methyl ester preparation, FA composition of PBMC was measured. We observed no effect of treatment on body weight gain, body condition score change, or wither height change. Heifers receiving the N3 diet had a lower temperature response to Pasteurella challenge compared with both the mix and N6 diets. Heifers consuming the N3 diet had a greater content of total n-3 FA, α-linolenic acid, and eicosapentaenoic acid in PBMC compared with heifers fed the N6 and mix diets. Heifers receiving the N3 diet also had a lower content of total n-6 FA, linoleic acid, and arachidonic acid in PBMC than heifers fed the N6 and mix diets. In conclusion, our study determined that feeding weaned female Holstein heifers a diet high in n-3 FA increased concentrations of n-3 FA in PBMC.