Determination Of Plasma Concentrations Research Articles

Serum Level of Metalloproteinase-2 but not Metalloproteinase-9 Rises in Patients With Failed Back Surgery Syndrome After Spinal Cord Stimulation

The impact of spinal cord stimulation (SCS) on serum levels of metalloproteinase-2 (MMP-2) and metalloproteinase-9 (MMP-9) was assessed in a group of patients with failed back surgery syndrome (FBSS). The study was to give new insights into the SCS mechanism of action and the role of MMP-2 and MMP-9 in the development of NP. Clinical assessments were performed and biochemical markers were determined in two groups of patients: the control group (24 individuals) and the FBSS group (24 patients). Seventeen patients with the FBSS had SCS implanted and were examined before surgical procedure, one month after (17 patients), and three months after operation (12 patients). Clinical status was assessed with the use numeric rating scale, pain rating index of McGill pain questionnaire, Oswestry disability index and Beck depression inventory. MMP-2 and MMP-9 serum levels were determined using gelatin zymography. Immunoenzymatic method was employed to determine plasma concentrations of tissue inhibitors of metalloproteinases (TIMPs). Levels of MMP-2 and TIMP-2 were higher in the FBSS group compared to the control group. The difference was statistically significant (p < 0.001 and p = 0.004, respectively). The concentration of MMP-2 was significantly increased (p = 0.0135) one-month post-SCS and remained elevated but stable up to three months after implantation. TIMP-2, MMP-2/TIMP-2, MMP-9, TIMP-1, and MMP-9/TIMP-1 serum levels did not change significantly. MMPs may play a role in the development of FBSS. SCS increases the already elevated MMP-2 serum levels which are associated with neuroinflammatory processes in FBSS patients.

Melatonin-improved buffalo semen quality during nonbreeding season under tropical condition

The role of melatonin in protecting spermatozoa from different kinds of injury has been widely reported. The present study aimed to test whether treatment of buffalo bulls with melatonin could ameliorate sperm function during nonbreeding season under tropical condition. Ten Murrah buffalo bulls were randomly allocated into control and treated groups of equal numbers of bulls to study the effect of melatonin on semen characteristics, seminal plasma constituents, blood plasma hormonal levels, and antioxidant enzyme activities during nonbreeding season. Treated bulls were implanted with melatonin (18 mg/50 kg of body weight) for a period of 2 mo. During this period, semen was collected twice a week, and blood samples were collected weekly to determine plasma concentration of melatonin and LH and activities of antioxidant enzymes. During nonbreeding season, melatonin implantation improved semen characteristics by increasing (P < 0.05) percentages of sperm with forward motility, viability, total motile sperm, and rapid motility, average path, curvilinear, and straight-line velocity and amplitude of lateral head displacement and decreasing (P < 0.05) percentages of abnormal sperm and linearity index as compared to the control group. Furthermore, melatonin implantation increased (P < 0.05) seminal plasma concentrations of total protein, albumin, and cholesterol and decreased (P < 0.05) seminal plasma aspartate aminotransferase activity. In addition, melatonin-implanted bulls exhibited an increase (P < 0.05) in red blood cells superoxide dismutase activity compared to untreated bulls. In conclusion, melatonin implantation successfully improved semen quality of buffalo bulls during nonbreeding season under tropical condition.

Prediction of Circulating Adipokine Levels Based on Body Fat Compartments and Adipose Tissue Gene Expression

Background: Adipokines are hormones secreted from adipose tissue (AT), and a number of them have been established as risk factors for chronic diseases. However, it is not clear whether and to what extent adiposity, gene expression, and other factors determine their circulating levels. Objectives: To assess to what extent adiposity, as measured by the amount of subcutaneous AT (SAT) and visceral AT (VAT) using magnetic resonance imaging, and gene expression levels in SAT determine plasma concentrations of the adipokines adiponectin, leptin, soluble leptin receptor, resistin, interleukin 6, and fatty acid-binding protein 4 (FABP4). Methods: We performed a cross-sectional analysis of 156 participants from the EPIC Potsdam cohort study and analyzed multiple regression models and partial correlation coefficients. Results: For leptin and FABP4 concentrations, 81 and 45% variance were explained by SAT mass, VAT mass, and gene expression in SAT in multivariable regression models. For the remaining adipokines, AT mass and gene expression explained <16% variance of plasma concentrations. Gene expression in SAT was a less important predictor compared to AT mass. SAT mass was a better predictor than VAT mass for leptin (partial correlation r = 0.81, 95% confidence interval 0.75–0.86, vs. r = 0.58, 95% confidence interval 0.46–0.67), while differences between AT compartments were small for the other adipokines. Conclusions: While plasma levels of leptin and FABP4 can be explained in a large and medium part by the amount of AT and SAT gene expression, surprisingly, these predictors explained only little variance for all other investigated adipokines.

Quercetin, but Not Epicatechin, Decreases Plasma Concentrations of Methylglyoxal in Adults in a Randomized, Double-Blind, Placebo-Controlled, Crossover Trial with Pure Flavonoids

Methylglyoxal (MGO) is the most potent precursor of advanced glycation end products (AGEs). MGO and AGEs have been associated with diabetes, its complications, and other age-related diseases. Experimental studies have shown that the flavonoids quercetin and epicatechin are able to scavenge MGO and lower AGE formation. Data on the effects of these flavonoids on MGO and AGE concentrations in humans are not yet available. We therefore investigated the effect of quercetin and epicatechin on the concentrations of MGO and AGEs in a post hoc analysis. Thirty-seven apparently healthy, nonsmoking adults with a systolic blood pressure between 125 and 160 mm Hg at screening were included in a randomized, double-blind, placebo-controlled crossover trial. Participants ingested (-)-epicatechin (100 mg/d), quercetin 3-glucoside (160 mg/d), or placebo capsules for periods of 4 wk separated by 4-wk washout periods. Fasting blood samples were collected at the start and end of each intervention period. Liquid chromatography-tandem mass spectrometry was used to determine plasma concentrations of the dicarbonyl compounds MGO, glyoxal (GO), and 3-deoxyglucosone (3-DG) and free and protein-bound AGEs. Gene expression of glyoxalase 1 (GLO1), the enzyme involved in the degradation of MGO, was determined by either microarray or quantitative reverse transcriptase-polymerase chain reaction. The treatment effect (Δtreatment - Δplacebo) of quercetin on MGO was -40.2 nmol/L (95% CI: -73.6, -6.8nmol/L; P=0.019), a decrease of 11% from baseline values, whereas GO, 3-DG, and free and protein-bound AGEs did not change significantly. Epicatechin did not affect the concentrations of dicarbonyls and free and protein-bound AGEs. We did not find a significant change in expression of GLO1. In apparently healthy (pre)hypertensive men and women, quercetin but not epicatechin decreased plasma MGO concentrations. Quercetin may potentially form a new treatment strategy for diseases in which MGO plays a pivotal role. This study was registered at clinicaltrials.gov as NCT01691404.

Daily Consumption of Oregon Hazelnuts Affects α-Tocopherol Status in Healthy Older Adults: A Pre-Post Intervention Study

Inadequate vitamin E and magnesium intakes are of concern for older adults owing to the associated incidence of age-related diseases. This study was designed to determine the extent to which a 16-wk intervention with hazelnuts alters vitamin E and magnesium status in a group of older men and women, and used a pre-post intervention design without a control group to adjust for temporal changes. Participants (n=32 including 22 women; mean±SD age: 63±6 y) consumed hazelnuts (∼57 g/d) for 16 wk. Blood and urine samples and anthropomorphic measures were taken at the start and end of the intervention to determine plasma concentrations of α-tocopherol and serum concentrations of magnesium, lipids, glucose, insulin, and high-sensitivity C-reactive protein along with urinary vitamin E metabolites; several other micronutrients were measured by a lymphocyte proliferation assay. There were 3 primary endpoints, calculated as the mean changes in measurements between baseline and the end of the 16-wk intervention for 1) plasma α-tocopherol, 2) urinary α-carboxyethyl hydroxychromanol (α-CEHC; an α-tocopherol metabolite), and 3) serum magnesium. Hazelnut consumption increased concentrations of the urinary α-tocopherol metabolite α-CEHC (mean±SD: 0.84±0.45 to 1.14±0.50 µmol/g creatinine; P=0.0006). In addition, hazelnut consumption increased serum concentrations of magnesium (+2.1%, P=0.05), decreased concentrations of fasting glucose (-3.4%, P=0.03) and LDL cholesterol (-6.0%, P=0.02), and decreased total:HDL cholesterol ratios (-4.5%, P=0.009). No significant changes were observed in blood pressure, lymphocyte proliferation assays, and serum concentrations of insulin, high-sensitivity C-reactive protein, triglyceride, α-tocopherol, or HDL cholesterol. Consuming hazelnuts improves a biomarker of vitamin E status in older adults. Vitamin E is a shortfall micronutrient, as identified by the Dietary Guidelines for Americans 2015-2020, which frequently is consumed at levels less than the Estimated Average Requirement of 12 mg/d; thus, hazelnuts should be considered as part of a healthy dietary pattern. This trial was registered at clinicaltrials.gov as NCT03485989.

Investigation on pharmacochemistry and pharmacokinetics of atractylenolides from Atractylodes in vivo based on UPLC-MS combined with everted gut sac model in vitro

The article aimed to study intestinal absorption and pharmacokinetic characterization of three atractylenolides from low polar component of Atractylodes (LPA). LC-MS method was used to find absorbed components combined with pharmacochemistry and to determine plasma concentration combined with DAS 3.0 to calculate the PK parameters. Moreover, the everted gut sac model was used to investigate rat small intestinal absorption and transport characterization of atractylenolide I, II, III. In total, eight components were identified from the absorbed components, including two lactones (6, 7), three volatile oil (2, 3, 8), acids (1), prenylated dihydrobenzofuran derivative (4) and luteolin (5), respectively. The three atractylenolides are rapidly absorbed with Tmax as 1 h and more compatible with the two-compartment model in rats, and good linearity between Cmax and concentration. The cumulative absorption per unit area Q (ug·cm−2) of three atractylenolides were no significant difference and all linearly absorbed with different time, concentration in different intestinal segments, which indicated its absorption process was passive transport. In addition, the atractylenolides III showed most high absorption rate in all intestinal segments, and the absorption site of atractylenolides II was mainly duodenum segments. LC-MS is a systematically applicable approach for rapid screening, identification and quanlitation of absorbed components derived from atractylodes in vivo. Three atractylenolides showed similar PK characterization and passive transport through all intestinal segments. It looks more safe with less drug interaction.

Safety, tolerability, pharmacokinetics and pharmacokinetic-pharmacodynamic modelling of the novel H4 receptor inhibitor SENS-111 using a modified caloric test in healthy subjects.

A Phase 1 study was performed to evaluate safety, pharmacokinetics (PK) and pharmacodynamics (PD) of the selective histamine H4 receptor antagonist SENS-111, an oral small molecule. One hundred healthy subjects were randomized in a placebo-controlled, double-blind study evaluating single-ascending doses (SAD; 100-500mg) and multiple-ascending doses (MAD; 50-150mgday-1 , 4 days; 200-250mgday-1 , 7 days). Effects of SENS-111 on nystagmus and vertigo induced by modified caloric tests were measured in the MAD studies. Population PK and PK/PD models were developed using a nonlinear mixed-effects approach. SENS-111 was well tolerated with mild to moderate events. No sedation was reported. A maximal tolerated dose was not reached. Dose-proportional increases in concentrations were seen up to 200mg and more than dose-proportional thereafter, with mean half-life between 24 and 56h. The caloric test induced mild but measurable vertigo and nystagmus with large intra/inter-individual variation for all parameters. SENS-111 did not significantly impact nystagmus but significantly improved latency of vertigo appearance/disappearance, duration and European Evaluation of Vertigo questionnaire parameters vs. baseline. A two-compartment model with first-order absorption, distribution and elimination best fit the data. PK/PD indirect modelling applied to vertigo duration and latency of appearance indicated maximum activity between 100 and 500ngml-1 plasma concentrations, corresponding to 100 and 200mgday-1 , which are appropriate for clinical efficacy evaluations in vestibular diseases. SENS-111 is a well-tolerated first-in-class H4 receptor antagonist with acceptable PK for oral daily dosing. PK/PD modelling determined plasma concentrations and doses for efficacy studies in patients with vertigo symptoms.

Effect of γ-hydroxybutyrate (GHB) on driving as measured by a driving simulator.

Gamma-hydroxybutyrate acid (GHB), a GABAB receptor agonist approved for treatment of narcolepsy, impairs driving ability, but little is known about doses and plasma concentrations associated with impairment and time course of recovery. To assess effects of oral GHB (Xyrem®) upon driving as measured by a driving simulator, and to determine plasma concentrations associated with impairment and the time course of recovery. Randomized, double-blind, two-arm crossover study, during which 16 participants received GHB 50mg/kg orally or placebo. GHB blood samples were collected prior to and at 1, 3, and 6h post dosing. Driving simulator sessions occurred immediately after blood sampling. Plasma GHB was not detectable at baseline or 6h post dosing. Median GHB concentrations at 1 and 3h were 83.1mg/L (range 54-110) and 24.4mg/L (range 7.2-49.7), respectively. Compared to placebo, at 1h post GHB dosing, significant differences were seen for the life-threatening outcome collisions (p < 0.001) and off-road accidents (p = 0.018). Although driving was not faster, there was significantly more weaving and erratic driving with GHB as measured by speed deviation (p = 0.002) and lane position deviation (p = 0.004). No significant impairment regarding driving outcomes was found in the GHB group at 3 and 6h post dose. GHB in doses used to treat narcolepsy resulted in severe driving impairment at 1h post dosing. After 3 to 6h, there was full recovery indicating that safe driving is expected the next morning after bedtime therapeutic GHB use in the absence of other substances.

Pharmacokinetics of enrofloxacin and its metabolite ciprofloxacin following single-dose subcutaneous injection in black-tailed prairie dogs (Cynomys ludovicianus).

OBJECTIVE To determine plasma concentrations of enrofloxacin and its active metabolite ciprofloxacin following single-dose SC administration to black-tailed prairie dogs (Cynomys ludovicianus). ANIMALS 8 captive healthy 6-month-old sexually intact male black-tailed prairie dogs. PROCEDURES Enrofloxacin (20 mg/kg) was administered SC once to 6 prairie dogs and IV once to 2 prairie dogs. A blood sample was collected from each animal immediately before (0 hours) and 0.5, 1, 2, 4, 8, 12, and 24 hours after drug administration to evaluate the pharmacokinetics of enrofloxacin and ciprofloxacin. Plasma enrofloxacin and ciprofloxacin concentrations were quantified with ultraperformance liquid chromatography-mass spectrometry, and noncompartmental pharmacokinetic analysis was performed. RESULTS Enrofloxacin was biotransformed to ciprofloxacin in the prairie dogs used in the study. For total fluoroquinolones (enrofloxacin and ciprofloxacin), the mean (range) of peak plasma concentration, time to maximum plasma concentration, and terminal half-life after SC administration were 4.90 μg/mL (3.44 to 6.08 μg/mL), 1.59 hours (0.5 to 2.00 hours), and 4.63 hours (4.02 to 5.20 hours), respectively. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that administration of enrofloxacin (20 mg/kg, SC, q 24 h) in black-tailed prairie dogs may be appropriate for treatment of infections with bacteria for which the minimum inhibitory concentration of enrofloxacin is ≤ 0.5 μg/mL. However, clinical studies are needed to determine efficacy of such enrofloxacin treatment.

Plasma concentrations of lidocaine following laryngeal administration or laryngeal and intratesticular administration in cats.

OBJECTIVE To determine plasma concentrations of lidocaine after laryngeal administration or laryngeal and intratesticular administration in cats. ANIMALS 14 healthy adult sexually intact male cats (7 cats/treatment). PROCEDURES Cats were randomly allocated to receive 0.1 mL of 2% or 10% lidocaine hydrochloride solution (treatments L2 and L10, respectively) sprayed on the larynx for laryngeal desensitization, followed by endotracheal intubation and isoflurane anesthesia. After a 7-day washout period, cats were again randomly allocated to receive treatment L2 or L10, and castration was performed under isoflurane anesthesia following intratesticular administration of 2% lidocaine solution (0.1 mL/kg). In both experiments, a blood sample for measurement of plasma lidocaine concentration was obtained before (0 minutes) and 3, 5, 10, 15, 20, 30, 45, 60, and 75 minutes after laryngeal administration of lidocaine solution. Anesthesia was discontinued at 60 minutes. Plasma lidocaine concentrations were measured with high-performance liquid chromatography. RESULTS After treatments L2 and L10, median maximum plasma lidocaine concentrations were 34.1 ng/mL (range, 0 to 279.4 ng/mL) and 93.6 ng/mL (range, 79.3 to 182.2 ng/mL), respectively. Time to maximum plasma concentration was 10 minutes (range, 0 to 20 minutes) for each treatment. When cats received intratesticular lidocaine administration following L2 or L10 treatment, median maximum plasma concentration was 181.0 ng/mL (range, 103.7 to 600.2 ng/mL) and 301.2 ng/mL (range, 265.8 to 1,770.0 ng/mL), respectively. CONCLUSIONS AND CLINICAL RELEVANCE On the basis of these data, combined laryngeal and intratesticular administration of lidocaine solution at a total dose of approximately 5 mg/kg appears to be safe for use in healthy adult cats.

LC-MS-MS Determination of Oxcarbazepine and an Active Metabolite in Human Plasma for Clinical Application.

A bioanalytical method for the simultaneous determination of oxcarbazepine (OXC) and its pharmacologically active metabolite, 10, 11-dihydro-10-hydroxycarbamazepine (HOXC), in human plasma was developed using a high-performance liquid chromatography with tandem mass spectrometry. After protein precipitation by acetonitrile, the analytes (OXC and HOXC) and a stable-labeled isotope of OXC as an internal standard (IS) were chromatographed on a Synergi Hydro-RP column (2.0 mm × 50 mm, 4 μm) with a gradient elution at a flow rate 0.5 mL/min. Detection was performed in electrospray ionization in the positive mode by monitoring the selected ion transitions at m/z 253.1 → 180.2, m/z 255.1 → 192.2 and m/z 257.2 → 184.2 for OXC, HOXC and the IS, respectively. The method was validated according to current bioanalytical method validation guidelines. The calibration standard curve ranged from 0.02 to 10 μg/mL for OXC and 0.1-50 μg/mL for HOXC using only 0.05 mL of plasma. No interferences were detected in blank plasma and hemolyzed plasma did not have any impacts on the assay. Accuracy and precision in the intra- and inter-batch reproducibility were within 15%. Neither cross-analytes inter-conversion nor matrix effects were observed. The method was successfully applied to determine plasma concentrations of OXC and HOXC to support a clinical study.

Development and Validation of a Bioanalytical Method to Quantitate Enzalutamide and its Active Metabolite N-Desmethylenzalutamide in Human Plasma: Application to Clinical Management of Patients With Metastatic Castration-Resistant Prostate Cancer.

Enzalutamide is a potent androgen-signaling receptor inhibitor and is licensed for the treatment of metastatic castration-resistant prostate cancer. N-desmethylenzalutamide is the active metabolite of enzalutamide. A method to quantitate enzalutamide and its active metabolite was developed and validated according to the European Medicine Agency guidelines. Enzalutamide and N-desmethylenzalutamide were extracted by protein precipitation, separated on a C18 column with gradient elution and analyzed with tandem quadrupole mass spectrometry in positive ion mode. A stable deuterated isotope (D6-enzalutamide) was used as an internal standard. The method was tested and stability was studied in real-life patients with metastatic castration-resistant prostate cancer patients treated with enzalutamide. The calibration curve covered the range of 500-50,000 ng/mL. Within- and between-day precisions were <8% and accuracies were within 108% for both enzalutamide and N-desmethylenzalutamide. Precisions for lower limit of quantification level were <10% and accuracies within 116% for enzalutamide and N-desmethylenzalutamide. Enzalutamide and N-desmethylenzalutamide stability was proven for 24 hours for whole blood at ambient temperature and 23 days for plasma at both ambient temperature and 2-8°C. Long-term patient plasma stability was shown for 14 months at -40°C. This bioanalytical method was successfully validated and applied to determine plasma concentrations of enzalutamide and N-desmethylenzalutamide in clinical studies and in routine patient care.

Plasma concentrations of alpha-1-acid glycoprotein in preterm and term newborns: influence of mode of delivery and implications for plasma protein binding of local anaesthetics

BackgroundAlpha-1-acid glycoprotein (AAGP) is an acute-phase protein with high affinity for amide local anaesthetics (LAs), and a major determinant of free and potentially toxic concentrations of LAs in plasma. Neonates are known to have lower plasma concentrations of AAGP than adults, and are at risk of developing high free concentrations of LAs. Data regarding AAGP in newborns are so far sparse. The aim of this study was to determine plasma concentrations of AAGP after delivery of preterm and term infants, and to investigate correlations between AAGP and gestational age, birth weight, gender, and mode of delivery. MethodsIn this prospective observational study, blood was sampled from umbilical cords of 70 newborn infants born at gestational weeks 27–42 immediately after delivery. Blood samples were subsequently analysed for AAGP plasma concentrations with an immunoturbidimetric assay. ResultsWe found higher concentrations of AAGP in infants born vaginally compared with those who were delivered by elective Caesarean section [median (inter-quartile range) 0.189 g litre−1 (0.142–0.263 g litre−1) vs 0.110 g litre−1 (0.094–0.157 g litre−1; P=0.0003)], respectively. There was a correlation between gestational age and AAGP concentrations (r=0.50; P=0.011), with significantly higher concentrations in the more mature infants. Gender and birth weight did not appear to influence the plasma concentrations of AAGP. ConclusionsAlpha-1-acid glycoprotein concentrations in newborns are influenced both by gestational age and mode of delivery. Thus, when dosing local anaesthetics in a parturient, these factors should be taken into account.

Mechanistic study for the simultaneous determination of metformin and teneligliptin in human plasma using hydrophilic interaction liquid chromatography-MS/MS.

A simple, selective and sensitive hydrophilic interaction liquid chromatography-MS/MS method is developed for the simultaneous determination of metformin (MET) and teneligliptin (TEN) in human plasma using deuterated internal standards. The mechanism of retention of analytes was studied by varying the proportion of organic diluent, buffer strength, pH of the mobile phase and temperature. The results showed a mixed-mode mechanism comprising of hydrophilic (partition) and electrostatic interaction (ion exchange) for MET and essentially hydrophilic for TEN. The linear calibration curves were established in the concentration range of 1.0-1000ng/ml for MET and 0.50-750ng/ml for TEN. The method was applied to determine plasma concentration of MET and TEN in healthy subjects.

Determination of telbivudine in the plasma of chronic hepatitis B patients in long-term treatment by high-performance liquid chromatographic-tandem mass spectrometry.

Creatine kinase elevation is commonly reported in telbivudine-treated patients. However, little is known about the relationship between this adverse drug reaction and plasma concentration. In this study, a sensitive, rapid and safe quantitative bioanalytical method has been established by using LC-MS/MS for the determination of telbivudine in a clinical study of chronic hepatitis B patients. The assay was linear in a dynamic 10-10,000 ng/mL range (r2 > 0.999) and total analysis time was 6 min in this method. The validated method was applied to quantitatively determine plasma concentration in chronic hepatitis B patients during long-term telbivudine treatment. The results revealed that telbivudine concentration in the creatine kinase-elevated group (707.92-2788.78 ng/mL) was significantly higher than those with normal creatine kinase (412.63-1108.32 ng/mL). This method was adapted for therapeutic drug monitoring.

Optimized plasma preparation is essential to monitor platelet-stored molecules in humans.

Platelets store a plethora of different molecules within their granules, modulating numerous pathways, not only in coagulation, but also in angiogenesis, wound healing, and inflammatory diseases. These molecules get rapidly released upon activation and therefore represent an easily accessible indirect marker for platelet activation. Accurate analysis of platelet-derived molecules in the plasma requires appropriate anticoagulation to avoid in vitro activation and subsequent degranulation of platelets, potentially causing artificially high levels and masking biologically relevant differences within translational research studies. However, there is still enormous heterogeneity among anticoagulants used to prevent unwanted platelet activation, so that plasma levels reported for platelet granule contents range over several orders of magnitude. To address this problem and to define the most robust method of plasma preparation to avoid in vitro platelet activation during processing, we compared plasma concentrations of the three platelet-stored factors thrombospondin (TSP-1), platelet factor 4 (PF4), and soluble P-selectin (sCD62P) between human blood samples anticoagulated with either citrate-theophylline-adenosine-dipyridamole (CTAD), acid-citrate-dextrose (ACD), citrate, ethylenediaminetetraacetic acid (EDTA) or heparin. Additionally, we assessed the effect of storage temperature and time between blood drawing and sample processing within the differentially anticoagulated samples. Our data strongly support the use of CTAD as anticoagulant for determining plasma concentrations of platelet-stored molecules, as anticoagulation with heparin or EDTA led to a 12.4- or 8.3-fold increase in plasma levels of PF4, respectively. Whereas ACD was similar effective as CTAD, citrate only showed comparable PF4 plasma levels when plasma was kept at 4°C. Moreover, blood sampling with CTAD as anticoagulant resulted in the most reproducible values, even when samples were processed at ambient temperature or after storage over 6 hours. In the latter case, anticoagulation with heparin or EDTA led to artificially high plasma levels indicative of in vitro platelet activation. Therefore, we want to raise scientific awareness for choosing CTAD as optimal anticoagulant for the detection of platelet-stored molecules in plasma.

Expression of two glycoprotein hormone receptors in larval, parasitic phase, and adult sea lampreys

All jawed vertebrates have three canonical glycoprotein hormones (GpHs: luteinizing hormone, LH; follicle stimulating hormone, FSH; and thyroid stimulating hormone, TSH) with three corresponding GpH receptors (GpH-Rs: LH-R, FSH-R, and TSH-R). In contrast, we propose that the jawless vertebrate, the sea lamprey (Petromyzon marinus), only has two pituitary glycoprotein hormones, lamprey (l)GpH and l-thyrostimulin, and two functional glycoprotein receptors, lGpH-R I and II. It is not known at this time whether there is a specific receptor for lGpH and l-thyrostimulin, or if both GpHs can differentially activate the lGpH-Rs. In this report, we determined the RNA expression of lGpH-R I and II in the gonads and thyroids of larval, parasitic phase, and adult lampreys. A highly sensitive dual-label fluorescent in situ hybridization technique (RNAScope™) showed lGpH-R I expression in the ovaries of larval lamprey, and co-localization and co-expression of lGpH-R I and II in the ovaries of parasitic phase and adult lampreys. Both receptors were also highly co-localized and co-expressed in the endostyle of larval lamprey and thyroid follicles of parasitic and adult lampreys. In addition, we performed in vivo studies to determine the actions of lamprey gonadotropin releasing hormones (lGnRHs) on lGpH-R I and II expression by real time PCR, and determined plasma concentrations of estradiol and thyroxine. Administration of lGnRH-III significantly (p ≤ 0.01) increased lGpHR II expression in the thyroid follicles of adult female lampreys but did not cause a significant increase in RNA expression of lGpH-R I and II in ovaries. Concomitantly, there was a significant increase (p ≤ 0.01) of plasma estradiol without any significant changes of plasma thyroxine concentrations in response to treatment to lGnRH-I, -II, or -III. In summary, our results provide supporting evidence that the lamprey pituitary glycoprotein hormones may differentially activate the lamprey GpH-Rs in regulating both thyroid and gonadal activities during each of the three life stages of the sea lamprey.

Lowering rumen-degradable and rumen-undegradable protein improved amino acid metabolism and energy utilization in lactating dairy cows exposed to heat stress

The objective of this study was to evaluate the effects of reducing dietary rumen-degradable protein (RDP) and rumen-undegradable protein (RUP) on protein and energy metabolism in heat-stressed dairy cows. Eighteen primiparous and 30 multiparous mid-lactation Holstein cows were used in a completely randomized design arranged in a 2 × 2 factorial (n = 12/treatment). Cows were randomly assigned to 1 of 4 dietary treatments that included 2 levels of RDP (10 and 8%; D) and 2 levels of RUP (8 and 6%; U) of dry matter for 21 d as (1) 10D:8U, (2) 8D:8U, (3) 10D:6U, and (4) 8D:6U. Diets were isoenergetic and contained 50% forage and 50% concentrate (dry matter basis). Cows were housed in a freestall barn. Three weeks before start of treatments, all animals were fed the 10D:8U diet and received supplemental cooling to prevent heat stress. During the treatment period, cows experienced a daily increment in temperature-humidity index from 74 to 82 for 1000 to 2000 h. Blood samples were collected on d -1 and 21 of the treatment period to determine plasma concentrations of AA, glucose, insulin, fatty acids, and β-hydroxybutyrate. For primiparous cows, reducing from 10 to 8% RDP decreased insulin concentrations. For multiparous cows, we found significant RDP by RUP interactions for insulin, β-hydroxybutyrate, fatty acids, total essential AA, and 3-methylhistidine concentrations. Reducing from 10 to 8% RDP decreased insulin concentrations at 6% RUP, but concentrations did not change when reducing RDP at 8% RUP. Reducing from 10 to 8% RDP decreased β-hydroxybutyrate concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 10 to 8% RDP increased nonesterified fatty acid and total essential AA concentrations at 8% RUP, but concentrations did not change when reducing RDP at 6% RUP. Reducing from 8 to 6% RUP decreased 3-methylhistidine concentration at 8% RDP, but not at 10% RDP. Reducing from 8 to 6% RUP increased milk protein yield efficiency in primiparous and multiparous cows. These results indicate that reducing RDP and RUP lowers circulating insulin, which was associated with mobilization and utilization of fatty acids. Reduced RDP and RUP increases the use of AA to maintain milk protein synthesis and limit AA catabolism in cows exposed to warm climates.

Treatment-Induced Viral Cure of Hepatitis C Virus-Infected Patients Involves a Dynamic Interplay among three Important Molecular Players in Lipid Homeostasis: Circulating microRNA (miR)-24, miR-223, and Proprotein Convertase Subtilisin/Kexin Type 9

In patients with chronic hepatitis C virus (HCV) infection, viral hijacking of the host-cell biosynthetic pathways is associated with altered lipid metabolism, which contributes to disease progression and may influence antiviral response. We investigated the molecular interplay among four key regulators of lipid homeostasis [microRNA (miR)-122, miR-24, miR-223, and proprotein convertase subtilisin/kexin type 9 (PCSK9)] in HCV-infected patients (n=72) who achieved a treatment-based viral cure after interferon-based therapy with first-generation direct-acting antivirals. Real-time PCR was used to quantify microRNA plasma levels, and ELISA assays were used to determine plasma concentrations of PCSK9. We report that levels of miR-24 and miR-223 significantly increased in patients achieving sustained virologic response (SVR), whereas the levels of miR-122, a liver-specific cofactor for HCV infection, decreased in these patients. PCSK9 concentrations were significantly increased in SVRs, suggesting that PCSK9 may help impede viral infection. The modulatory effect of PCSK9 on HCV infection was also demonstrated in the context of HCV-infected Huh-7.5.1 cells employing recombinant human PCSK9 mutants. Together, these results provide insights into a novel coordinated interplay among three important molecular players in lipid homeostasis — circulating miR-24, miR-223 and PCSK9 — whose regulation is affected by HCV infection and treatment-based viral cure.

Schisandra chinensis extract decreases chloroacetaldehyde production in rats and attenuates cyclophosphamide toxicity in liver, kidney and brain

Ethnopharmacological relevanceSchisandra chinensis (Turcz.) Baill (S. chinensis) has been used for thousands years in China, and is usually applied in treatment of urinary tract disorders and liver injury. S. chinensis extract (SCE) has board protective effects on liver, kidney and nervous system. Schisandra lignans are generally considered as the bioactive components of SCE. Aim of the studyTo investigate the pharmacokinetic herb-drug interactions (HDIs) between SCE and cyclophosphamide (CTX). To evaluate the protective effects of SCE against CTX induced damage in rat liver, kidney and brain. Materials and methodsThe pharmacokinetic HDIs between SCE and CTX were investigated by determining plasma concentrations of CTX and three metabolites, namely 4-ketocyclophosphamide (4-Keto), 2-dechloroethylcyclophosphamide (DCCTX) and carboxyphosphamide (CPM) using a previously developed UPLC-MS/MS method. To evaluate the protective effects of SCE pretreatment, toxicity and oxidation stress assessments along with histology investigations were carried out in rat liver, kidney and brain. ResultsThe equimolar produced metabolite DCCTX was chosen to reflect chloroacetaldehyde (CAA, a toxic metabolite of CTX) production in rats. Single-dose pretreatment of SCE significantly reduced CAA production and decreased the Cmax and AUC0–24h of DCCTX by 69% and 49% respectively (P < 0.05). After pretreated with SCE for 7 consecutive days, the Cmax and AUC0–24h of DCCTX were still decreased (−25% and −37%, P < 0.05) when compared with CTX alone group. Parallel toxicity and oxidation stress investigations showed that single-dose SCE pretreatment significantly decreased plasma BUN and Cr levels (−12% and −46%, respectively) and reduced liver AST activity (−32%). Moreover, SCE pretreatment potently increased the brain GSH content by 7.8-fold, and reduced MDA levels in rat liver, kidney and brain by 39%, 28% and 31%, respectively (compared with CTX alone group). The protective effects of SCE were also supported by histological observations. ConclusionOur experiment results suggest that S. chinensis may find use as a complementary medicine in CTX treatment.