Development and validation of a bioanalytical assay for the measurement of total and unbound teicoplanin in human serum.

BACKGROUND: The glycopeptide teicoplanin is considered first-line treatment for severe infections caused by Gram-positive bacteria. Individualized treatment of teicoplanin is gaining interest. As only protein-unbound drug is pharmacologically active, a sensitive assay measuring unbound and total teicoplanin is indispensable for pharmacological research and dose optimization. OBJECTIVES: To develop and validate a UPLC-MS/MS method to quantify unbound and total teicoplanin in human serum. METHODS: The developed assay was validated according to the ICH guideline M10 on Bioanalytical Method Validation and study sample analysis. Unbound teicoplanin was obtained by ultrafiltration. The assay was cross-validated with a quantitative microsphere (QMS) immunoassay in a side-by-side comparison using 40 patient samples. RESULTS: With the developed and validated method, all main teicoplanin components (A2-1, A2-2/A2-3, A2-4/A2-5 and A3-1) can be quantified. Total run time was 5.5 min. Concentration range was 2.5-150 mg/L for total and 0.1-25 mg/L for unbound teicoplanin. Precision (coefficient of variation) and accuracy (bias) of total teicoplanin were 5.97% and 107%, respectively, and 7.17% and 108%, respectively, for unbound teicoplanin.Bland-Altman analysis showed total concentrations measured with the UPLC-MS/MS method were equivalent to the results of the QMS immunoassay. A total of 188 samples from 30 patients admitted to the ICU and haematology department were measured; total concentrations ranged between 2.92 and 98.5 mg/L, and unbound concentrations ranged between 0.37 and 30.7 mg/L. CONCLUSIONS: The developed method provided rapid, precise and accurate measurement of unbound and total teicoplanin. The developed method is now routinely applied in pharmacological research and clinical practice.

Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Analysis of current perioperative management with Haemate® P/Humate P® in von Willebrand disease: Identifying the need for personalized treatment.

INTRODUCTION: Patients with Von Willebrand disease (VWD) are regularly treated with VWF-containing concentrates in case of acute bleeding, trauma and dental or surgical procedures. AIM: In this multicentre retrospective study, current perioperative management with a von Willebrand factor (VWF)/Factor VIII (FVIII) concentrate (Haemate® P) in patients with VWD was evaluated. PATIENTS/METHODS: Patients with VWD undergoing minor or major surgery between 2000 and 2015, requiring treatment with a VWF/FVIII concentrate (Haemate® P), were included. Achieved VWF activity (VWF:Act) and FVIII during FVIII-based treatment regimens were compared to predefined target levels in national guidelines. RESULTS: In total, 103 patients with VWD (148 surgeries) were included: 54 type 1 (73 surgeries), 43 type 2 (67 surgeries) and 6 type 3 (8 surgeries). Overall, treatment resulted in high VWF:Act and FVIII levels, defined as ≥0.20 IU/mL above predefined levels. In patients with type 1 VWD, respectively, 65% and 91% of trough VWF:Act and FVIII levels were higher than target levels. In patients with type 2 and type 3 VWD, respectively, 53% and 57% of trough VWF:Act and 72% and 73% of trough FVIII levels were higher than target level. Furthermore, FVIII accumulation over time was observed, while VWF:Act showed a declining trend, leading to significantly higher levels of FVIII than VWF:Act. CONCLUSION: High VWF:Act and accumulation of FVIII were observed after perioperative FVIII-based replacement therapy in patients with VWD, both underlining the necessity of personalization of dosing regimens to optimize perioperative treatment.

Tamoxifen dose and serum concentrations of tamoxifen and six of its metabolites in routine clinical outpatient care.

A sensitive and selective HPLC-MS/MS assay was used to analyze steady-state serum concentrations of tamoxifen, N-desmethyltamoxifen (E)-endoxifen, (Z)-endoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen, and 4'-hydroxytamoxifen to support therapeutic drug monitoring (TDM) in patients treated with tamoxifen according to standard of care. When the (Z)-endoxifen serum concentration was below the predefined therapeutic threshold concentration of 5.9 ng/mL, the clinician was advised to increase the tamoxifen dose and to collect another serum sample. Paired serum samples from patients at one dose level at different time points during the tamoxifen treatment were used to assess the intra-patient variability. A total of 251 serum samples were analyzed, obtained from 205 patients. Of these patients, 197 used 20 mg tamoxifen per day and 8 patients used 10 mg/day. There was wide variability in tamoxifen and metabolite concentrations within the dosing groups. The threshold concentration for (Z)-endoxifen was reached in one patient (12 %) in the 10 mg group, in 153 patients (78 %) in the 20 mg group, and in 26 (96 %) of the patients who received a dose increase to 30 or 40 mg/day. Dose increase from 20 to 30 or 40 mg per day resulted in a significant increase in the mean serum concentrations of all analytes (p < 0.001). The mean intra-patient variability was between 10 and 20 % for all analytes. These results support the suitability of TDM for optimizing the tamoxifen treatment. It is shown that tamoxifen dose is related to (Z)-endoxifen exposure and increasing this dose leads to a higher serum concentration of tamoxifen and its metabolites. The low intra-patient variability suggests that only one serum sample is needed for TDM, making this a relatively noninvasive way to optimize the patient's treatment.

Use of dried blood spots for the determination of serum concentrations of tamoxifen and endoxifen.

The anti-estrogenic effect of tamoxifen is suggested to be mainly attributable to its metabolite (Z)-endoxifen, and a minimum therapeutic threshold for (Z)-endoxifen in serum has been proposed. The objective of this research was to establish the relationship between dried blood spot (DBS) and serum concentrations of tamoxifen and (Z)-endoxifen to allow the use of DBS sampling, a simple and patient-friendly alternative to venous sampling, in clinical practice. Paired DBS and serum samples were obtained from 50 patients using tamoxifen and analyzed using HPLC-MS/MS. Serum concentrations were calculated from DBS concentrations using the formula calculated serum concentration = DBS concentration/([1-haematocrit (Hct)] + blood cell-to-serum ratio × Hct). The blood cell-to-serum ratio was determined ex vivo by incubating a batch of whole blood spiked with both analytes. The average Hct for female adults was imputed as a fixed value. Calculated and analyzed serum concentrations were compared using weighted Deming regression. Weighted Deming regression analysis comparing 44 matching pairs of DBS and serum samples showed a proportional bias for both analytes. Serum concentrations were calculated using [Tamoxifen] serum, calculated  = [Tamoxifen] DBS /0.779 and [(Z)-Endoxifen] serum, calculated = [(Z)-Endoxifen] DBS /0.663. Calculated serum concentrations were within 20 % of analyzed serum concentrations in 84 and 100 % of patient samples for tamoxifen and (Z)-endoxifen, respectively. In conclusion, DBS concentrations of tamoxifen and (Z)-endoxifen were equal to serum concentrations after correction for Hct and blood cell-to-serum ratio. DBS sampling can be used in clinical practice.

A prospective case series of women with estrogen receptor-positive breast cancer: levels of tamoxifen metabolites in controlled ovarian stimulation with high-dose tamoxifen.

Controlled ovarian stimulation (COS) in women with estrogen receptor (ER)-positive breast cancer is potentially harmful because of the increase in serum estrogen levels. During COS for cryopreservation of oocytes or embryos, these women may receive high doses of tamoxifen (60 mg) to modulate the ER and prevent extra growth of estrogen responsive tumours during COS. However, it is unknown whether adequate serum concentrations of endoxifen, the most important metabolite of tamoxifen, can be reached. The aim of this study is to evaluate whether the tamoxifen dose used in a tamoxifen-COS combined schedule for women with ER-positive breast cancer is high enough to reach endoxifen levels that are considered therapeutically effective to inhibit breast cancer growth. The four women with ER-positive breast cancer who underwent COS for cryopreservation of oocytes were prospectively studied at the Academic Medical Centre, Amsterdam, the Netherlands. Throughout COS, blood samples were collected and tamoxifen and endoxifen levels were determined by a validated high-performance liquid chromatography tandem mass spectrometry assay. The four women with ER-positive breast cancer underwent a total of five COS cycles, while additionally using tamoxifen 60 mg daily. The tamoxifen and endoxifen levels showed a large variability between the women, with endoxifen levels during the whole period of ovarian stimulation varying between 3.96 and 41.0 ng/ml. The average number of vitrified oocytes was 11 (5-14). Therapeutically effective endoxifen serum levels can be reached when tamoxifen is used to counteract estrogen levels during COS for fertility preservation, but not in all women. Large variations of tamoxifen and endoxifen levels between the women were observed.

Importance of highly selective LC-MS/MS analysis for the accurate quantification of tamoxifen and its metabolites: focus on endoxifen and 4-hydroxytamoxifen.

The antiestrogenic effect of tamoxifen is mainly attributable to the active metabolites endoxifen and 4-hydroxytamoxifen. This effect is assumed to be concentration-dependent and therefore quantitative analysis of tamoxifen and metabolites for clinical studies and therapeutic drug monitoring is increasing. We investigated the large discrepancies in reported mean endoxifen and 4-hydroxytamoxifen concentrations. Two published LC-MS/MS methods are used to analyse a set of 75 serum samples from patients treated with tamoxifen. The method from Teunissen et al. (J Chrom B, 879:1677-1685, 2011) separates endoxifen and 4-hydroxytamoxifen from other tamoxifen metabolites with similar masses and fragmentation patterns. The second method, published by Gjerde et al. (J Chrom A, 1082:6-14, 2005) however lacks selectivity, resulting in a factor 2-3 overestimation of the endoxifen and 4-hydroxytamoxifen levels, respectively. We emphasize the use of highly selective LC-MS/MS methods for the quantification of tamoxifen and its metabolites in biological samples.

[Intermediate-high risk pulmonary embolism: identification and treatment]. / Longembolie met intermediair-hoog risico op overlijden.

Patients with intermediate-high risk pulmonary embolism have a different mix of clinical symptoms. Optimal treatment of patients with intermediate high-risk pulmonary embolism is necessary to prevent short-term mortality. According to the current guidelines, the use of standard coagulation is the treatment of choice in hemodynamic stable patients with intermediate-high risk pulmonary embolism. Systemic thrombolytic therapy is recommended in patients with intermediate-high risk pulmonary embolism who circulatory deteriorate or who did not respond appropriately to standard anticoagulation. Catheter-guided thrombolysis is reserved for patients with intermediate-high risk pulmonary embolism who have a contraindication for systemic thrombolysis or did not respond to systemic thrombolysis. The timing and choice for the right treatment are significant treatment dilemmas. The development of pulmonary embolism response teams helps in the decision-making in patients with intermediate high-risk pulmonary embolism.

Stress-controlled decomposition routes in cubic AlCrN films assessed by in-situ high-temperature high-energy grazing incidence transmission X-ray diffraction.

The dependence of decomposition routes on intrinsic microstructure and stress in nanocrystalline transition metal nitrides is not yet fully understood. In this contribution, three Al0.7Cr0.3N thin films with residual stress magnitudes of -3510, -4660 and -5930 MPa in the as-deposited state were in-situ characterized in the range of 25-1100 °C using in-situ synchrotron high-temperature high-energy grazing-incidence-transmission X-ray diffraction and temperature evolutions of phases, coefficients of thermal expansion, structural defects, texture as well as residual, thermal and intrinsic stresses were evaluated. The multi-parameter experimental data indicate a complex intrinsic stress and phase changes governed by a microstructure recovery and phase transformations taking place above the deposition temperature. Though the decomposition temperatures of metastable cubic Al0.7Cr0.3N phase in the range of 698-914 °C are inversely proportional to the magnitudes of deposition temperatures, the decomposition process itself starts at the same stress level of ~-4300 MPa in all three films. This phenomenon indicates that the particular compressive stress level functions as an energy threshold at which the diffusion driven formation of hexagonal Al(Cr)N phase is initiated, provided sufficient temperature is applied. In summary, the unique synchrotron experimental setup indicated that residual stresses play a decisive role in the decomposition routes of nanocrystalline transition metal nitrides.

Formulation performance and processability window for manufacturing a dual-polymer amorphous solid dispersion via hot-melt extrusion and strand pelletization.

This work evaluates several compositions of an amorphous solid dispersion (ASD) comprising nimodipine (NMD) as poorly soluble model API in a dual-polymer carrier system. HPMC E5 and Eudragit E were used for the two polymeric carriers. The formulation was designed for hot-melt extrusion (HME) and subsequent strand pelletization. The aim was to identify a formulation window with desired functional ASD performance, i.e. physical stability and immediate API release, as well as processability in strand pelletization. Samples were prepared using small-scale methods, such as vacuum compression molding (VCM) and benchtop extrusion. Miscibility and phase studies were performed for a wide range of polymer ratios and three levels of API content (10-30% w/w). Ternary ASD formulations were phase-separated, yet physically stable upon exposure to elevated temperature/humidity. A study of phase composition showed that the drug molecules were predominantly solubilized in the Eudragit E fraction of the formulation. The miscibility study and Fourier-transform infrared spectroscopy indicated hydrogen (H)bond interactions between NMD and Eudragit E. In HPMC, the amorphous API was dispersed in polymeric matrix and stabilized due to anti-plasticization and the disruption of intermolecular Hbonding between API molecules. Concerning processability in strand pelletization the formulation is limited at high Eudragit E content. NMD and EE-rich phases exhibit low mixture glass transition, low melt stability and brittle breaking behavior upon strand cutting. The high viscosity and yield point of HPMC contributes to the mechanical robustness of the strand at temperatures relevant for processing. Formulation-intrinsic dissolution rates in VCM ASDs developed as an irregular function of polymer ratio, associated with diverse and competitive dissolution mechanisms in the polymers. With regard to the binary system of NMD with HPMC E5, surface crystallization was observed in VCM ASDs. For extruded pellets this was not the case, and a steady trend of formulation-intrinsic dissolution rate across different polymer ratios was observed. These discrepancies indicated a major influence of shear stress during sample preparation on HPMC-based ASD performance. Finally, a feasible formulation window within a polymer ratio of 1:2-2:3 Eudragit E:HPMC was identified in which Eudragit E acts as a dissolution rate enhancer and ASD stabilizer during dissolution.

Adhesive Cementation Promotes Higher Fatigue Resistance to Zirconia Crowns.

OBJECTIVE: The aim of this study was to investigate the influence of the cementation strategy on the fatigue resistance of zirconia crowns. The null hypothesis was that the cementation strategy would not affect the fatigue resistance of the crowns. METHODS AND MATERIALS: Seventy-five simplified molar tooth crown preparations were machined in glass fiber-filled epoxy resin. Zirconia crowns were designed (thickness=0.7 mm), milled by computer-aided design/computer-aided manufacturing, and sintered, as recommended. Crowns were cemented onto the resin preparations using five cementation strategies (n=15): ZP, luting with zinc phosphate cement; PN, luting with Panavia F resin cement; AL, air particle abrasion with alumina particles (125 µm) as the crown inner surface pretreatment + Panavia F; CJ, tribochemical silica coating as crown inner surface pretreatment + Panavia F; and GL, application of a thin layer of porcelain glaze followed by etching with hydrofluoric acid and silanization as crown inner surface pretreatment + Panavia F. Resin cement was activated for 30 seconds for each surface. Specimens were tested until fracture in a stepwise stress fatigue test (10,000 cycles in each step, 600 to 1400 N, frequency of 1.4 Hz). The mode of failure was analyzed by stereomicroscopy and scanning electron microscopy. Data were analyzed by Kaplan-Meier and Mantel-Cox (log rank) tests and a pairwise comparison (p<0.05) and by Weibull analysis. RESULTS: The CJ group had the highest load mean value for failure (1200 N), followed by the PN (1026 N), AL (1026 N), and GL (1013 N) groups, while the ZP group had the lowest mean value (706 N). Adhesively cemented groups (CJ, AL, PN, and GL) needed a higher number of cycles for failure than the group ZP did. The groups' Weibull moduli (CJ=5.9; AL=4.4; GL=3.9; PN=3.7; ZP=2.1) were different, considering the number of cycles for failure data. The predominant mode of failure was a fracture that initiated in the cement/zirconia layer. Finite element analysis showed the different stress distribution for the two models. CONCLUSION: Adhesive cementation of zirconia crowns improves fatigue resistance.

The influence of specimen attachment and dimension on microtensile strength.

The higher microtensile bond strength values found for specimens with a smaller cross-sectional area are often explained by the lower occurrence of internal defects and surface flaws. We hypothesized that this aberrant behavior is mainly caused by the lateral way of attachment of the specimens to the testing device, which makes the strength dependent on the thickness. This study showed that composite bars of 1x1x10, 1x2x10, and 1x3x10mm attached at their 1-mm-wide side (situation A) fractured at loads of the same magnitude, as a result of which the microtensile strength ( micro TS), calculated as F/A (force at fracture/cross-sectional area), significantly increased for specimens with decreasing thickness. Attachment at the 1-, 2-, or 3-mm-wide side (situation B) resulted in equal micro TS values (P > 0.05). Finite element analysis showed different stress patterns for situation A, but comparable patterns for situation B. Both situations showed the same maximum stress at fracture.

Dependence of the tray transmission factor on collimator setting and source-surface distance.

When blocks are placed on a tray in megavoltage x-ray beams, generally a single correction factor for the attenuation by the tray is applied for each photon beam quality. In this approach, the tray transmission factor is assumed to be independent of field size and source-surface distance (SSD). Analysis of a set of measurements performed in beams of 13 different linear accelerators demonstrates that there is, however, a slight variation of the tray transmission factor with field size and SSD. The tray factor changes about 1.5% for collimator settings varying between 4x4 cm and 40 x 40 cm for a 1 cm thick PMMA tray and approximately 3% for a 2 cm thick PMMA tray. The variation with field size is smaller if the source-surface distance is increased. The dependence on the collimator setting is not different, within the experimental uncertainty of about 0.5% (1 s.d.), for the nominal accelerating potentials and accelerator types applied in this study. It is shown that the variation of the tray transmission factor with field size and source-surface distance can easily be taken into account in the dose calculation by considering the volume of the irradiated tray material and the position of the tray in the beam. A relation is presented which can be used to calculate the numerical value of the tray transmission factor directly. These calculated values can be checked with only a few measurements using a cylindrical beam coaxial miniphantom.

Effect of electron contamination on scatter correction factors for photon beam dosimetry.

Physical quantities for use in megavoltage photon beam dose calculations which are defined at the depth of maximum absorbed dose are sensitive to electron contamination and are difficult to measure and to calculate. Recently, formalisms have therefore been presented to assess the dose using collimator and phantom scatter correction factors, Sc and Sp, defined at a reference depth of 10 cm. The data can be obtained from measurements at that depth in a miniphantom and in a full scatter phantom. Equations are presented that show the relation between these quantities and corresponding quantities obtained from measurements at the depth of the dose maximum. It is shown that conversion of Sc and Sp determined at a 10 cm depth to quantities defined at the dose maximum such as (normalized) peak scatter factor, (normalized) tissue-air ratio, and vice versa is not possible without quantitative knowledge of the electron contamination. The difference in Sc at dmax resulting from this electron contamination compared with Sc values obtained at a depth of 10 cm in a miniphantom has been determined as a multiplication factor, Scel, for a number of photon beams of different accelerator types. It is shown that Scel may vary up to 5%. Because in the new formalisms output factors are defined at a reference depth of 10 cm, they do not require Scel data. The use of Sc and Sp values, defined at a 10 cm depth, combined with relative depth-dose data or tissue-phantom ratios is therefore recommended. For a transition period the use of the equations provided in this article and Scel data might be required, for instance, if treatment planning systems apply Sc data normalized at d(max).

Magnetic resonance (MR) imaging of prostatic tumours, a comparison with X-ray CT and transrectal sonography (TRS).

A total of 7 healthy volunteers and 31 patients have been examined clinically, by MRI, TRS, and biopsy. In those patients with established carcinoma, a CT examination was also performed. For the MRI study, a superconducting MR 2000 imager (Picker International) operated at 0.15 T was used with multiplanar SE and IR sequences. SE sequences with long echo times detected prostatitis, adenoma and carcinoma of the prostate with a high degree of sensitivity. However, at present, differentiation between adenoma, prostatitis and carcinoma is not possible with sufficient accuracy. In these studies we were unable to establish a correlation between the signal pattern and staging and/or grading of the carcinoma. Reliable diagnosis of a prostate carcinoma still requires a biopsy. Because of the high soft tissue contrast and the possibility of selecting any orientation for the plane under investigation, however, MRI represents an improvement in the preoperative diagnosis of local spread.

[Results and complications of percutaneous nephropyelostomy in 215 patients]. / Ergebnisse und Komplikationen der perkutanen Nephropyelostomie bei 215 Patienten.

The results of 242 percutaneous nephropyelostomies in 215 patients were analysed retrospectively. No consideration was given to the experience of the individual operators. There was a total complication rate of 36.4% (mild complications 32.2%, serious complications 34.1%, fatality 0.4%). Rapid reduction in serum creatinine could be demonstrated following drainage in patients in whom renal function had been damaged by obstruction.

The influence of surface roughness on porcelain strength.

OBJECTIVES: In order to adjust occlusion, the functional surfaces of porcelain restorations are often ground and mechanical machining is even an essential part of the CAD-CAM process for these restorations. The aim of this study was to investigate the influence of the finishing procedures on the biaxial flexure strength of four commercial porcelains. METHODS: Four commercial porcelains of which two are used for metal-ceramic restorations (Flexo Ceram Dentine and Vita VM K68) and two for veneers and inlays (Duceram LFC Dentine and Cerinate BODY) are used in this study. For each porcelain, sixty discs (Ø = 22 mm, h = +/- 2.0 mm) were produced using twelve different finishing procedures. Twenty discs were left untreated, twenty discs were milled, using a high-speed diamond disc, and twenty discs were machined in a high-speed grinding/polishing device. Half of the samples were glazed. In each of these six groups, half of the samples were stored for 16 h at 80 degrees C in a 4% acetic acid solution. The biaxial flexure strength was determined using the ball-on-ring method. In each group the roughness of the surface was determined and examined via SEM. RESULTS: With the exception of Flexo Ceram Dentine, a significant correlation was found between the roughness of the surface and the biaxial strength: the smoother the surface, the stronger the sample. The differences in biaxial strength may be attributed to the stress concentration of an applied load due to the roughness of the surface caused by mechanical finishing or chemical action. The fact that the strength of Flexo Ceram Dentine was not affected by the different surface treatments is probably due to the size of the leucite particles, which apparently induce more stress concentration than the surface flaws and the roughness of the surface. SIGNIFICANCE: It was concluded that surface roughness determines the strength of a porcelain material, except where the inner structure of the material causes greater stress concentration than that caused by the combination of surface roughness and surface flaws.

Prevention of post-rape psychopathology: preliminary findings of a controlled acute rape treatment study.

Violent sexual assault such as rape typically results in extremely high levels of acute distress. The intensity of these acute psychological reactions may play a role in later recovery, with higher levels of immediate distress associated with poorer outcome. Unfortunately, post-rape forensic evidence collection procedures may serve to increase, rather than reduce initial distress, potentially exacerbating future psychopathology. To address these concerns, an acute time-frame hospital-based video intervention was developed to: (a) minimize anxiety during forensic rape exams, and (b) prevent post-rape posttraumatic stress disorder (PTSD), panic, and anxiety. Preliminary data indicated that (1) psychological distress at the time of the exam was strongly related to PTSD symptomatology 6 weeks post-rape, and (2) the video intervention successfully reduced distress during forensic exams.

Gene expression phenotypes for lipid metabolism and intramuscular fat in skeletal muscle of cattle.

Gene expression phenotypes were evaluated for intramuscular fat (IMF) in bovine skeletal muscle as an alternative to traditional estimates of IMF%. Gene expression data from a time course of LM development in high- and low-marbling Bos taurus cattle crosses were compared to identify genes involved in intramuscular adipocyte lipid metabolism with developmentally similar gene expression profiles. Three sets of genes were identified: triacylglyceride (TAG) synthesis and storage, fatty acid (FA) synthesis, and PPARγ-related genes. In an independent analysis in the LM of 48 Bos indicus cattle, TAG and FA gene sets were enriched in the top 100 genes of which expression was most correlated with IMF% (P = 1.2 × 10(-24) and 3.5 × 10(-9), respectively). In general, genes encoding enzymes involved in the synthesis of FA and TAG in the intramuscular adipocytes were present in the top 100 genes. In B. indicus, effects of a steroid hormone growth promotant (HGP), 2 experimental sites [New South Wales (NSW) and Western Australia (WA)], and 3 tenderness genotypes on the expression levels of genes in the TAG gene set and the correlation of gene expression with IMF% were investigated. Although correlation between expression of 12 individual TAG genes and IMF% was observed in HGP-treated animals in both experimental sites (mean r = 0.43), correlation was not observed for untreated animals at the NSW site (mean r = -0.07, P < 3 × 10(-6)). However, TAG genes showed an average 1.6-fold (P < 0.0004) reduction in expression in the LM of HGP-treated cattle relative to untreated cattle, an effect consistent across both experimental sites. Cattle possessing the favored tenderness calpain 1 and 3 and calpastatin alleles exhibited a greater (P = 0.008) reduction in expression in NSW (1.8-fold reduction, P = 0.0002) compared with WA (1.2-fold reduction, P = 0.03). Tenderness genotype had no impact (P > 0.05) on the correlation of TAG genes with IMF%. In general, the interactions among genotype, treatment and location, and TAG gene set gene expression were consistent with the interactions among the same factors and IMF% detected using 255 animals, of which the 48 in this study were a subset. Thus, the TAG gene set constitutes a gene expression phenotype able to predict effects of different genotypes and treatments on IMF% using much smaller groups than current approaches, even in animals with very low IMF%.

Development and validation of a quantitative assay for the determination of tamoxifen and its five main phase I metabolites in human serum using liquid chromatography coupled with tandem mass spectrometry.

A sensitive bioanalytical assay for the quantitative determination of tamoxifen and five of its phase I metabolites (N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen) in serum is described. The method has been fully validated at ranges covering steady-state serum concentrations in patients receiving therapeutic dosages of tamoxifen. The bioanalytical assay is based on reversed phase liquid chromatography coupled with tandem mass spectrometry in the positive ion mode using multiple reaction monitoring for drug (-metabolite) quantification. The sample pretreatment consists of protein precipitation with acetonitrile using only 50 µL of serum. In the past, numerous assays have been developed by other groups for the quantification of tamoxifen and its phase I metabolites. However, the number of metabolites included in these studies is very limited and only very few of these assays have been fully validated. A liquid chromatography tandem mass spectrometry assay for the quantification of tamoxifen and four phase I metabolites in human serum that was previously developed by our group is now explicitly improved and described herein. Time of analysis has been reduced by 50% and sensitivity was increased by a reduction of the lower limit of quantification from 1.0 to 0.2 ng/mL for 4-hydroxytamoxifen and 4'-hydroxytamoxifen. Additionally, two phase I metabolites that have never been quantified in human serum hitherto, namely 4'-hydroxytamoxifen and N-desmethyl-4'-hydroxytamoxifen, were included in this assay. Validation results demonstrate an accurate and precise quantification of tamoxifen, N-desmethyltamoxifen, N-desmethyl-4-hydroxytamoxifen, N-desmethyl-4'-hydroxytamoxifen, 4-hydroxytamoxifen and 4'-hydroxytamoxifen in human serum. The applicability of the assay was demonstrated and it is now successfully used to support clinical studies in which patient-specific dose optimization is performed based on serum concentrations of tamoxifen metabolites.