Simultaneous Quantification of Nine Antimicrobials by LC-MS/MS for Therapeutic Drug Monitoring in Critically Ill Patients.

BACKGROUND: Adequate antibiotic treatment is a prerequisite for the successful treatment of systemic infections. Based on accumulating scientific evidence, a fixed dosage regimen can lead to insufficient and ineffective antibiotic therapy. Thus, the aim of this study was to develop and validate a simplified, but sensitive method for the simultaneous quantification of antimicrobials by using liquid chromatography with tandem mass spectrometry (LC-MS/MS) for the development of personalized therapy regimens using therapeutic drug monitoring. METHODS: A method was developed for the simultaneous quantification of 9 antimicrobials (aciclovir, ampicillin, cefuroxime, ciprofloxacin, meropenem, metronidazole, piperacillin, rifampicin, and tazobactam) in lithium-heparin plasma. A simple sample preparation method and a chromatographic run time of 10 minutes enabled the quick processing of the samples. The method was validated according to the guidelines for bioanalytical method validation of the European Medicines Agency and addressed sensitivity, specificity, linearity, accuracy, precision, dilution integrity, carry-over, recovery, matrix effects, and stability. RESULTS: The chromatographic run time was 10 minutes and antimicrobials eluted at retention times ranging from 1.1 to 2.2 minutes. Calibration curve for all antimicrobials was linear over a range of 1-100 mg/L, and a 2-fold or 5-fold dilution of the samples was possible. The method accuracy ranged from 85.1% to 114.9% for all measured antimicrobials, and the within- and between-run precision values were <11.9% and <16.5% for the lower limit of quantification. No interferences and carry-over were observed. The samples were stable for at least 5 hours at room temperature or in the autosampler (10°C). CONCLUSIONS: The LC-MS/MS method developed in this study is appropriate and practical for the therapeutic drug monitoring of antimicrobials in the daily clinical laboratory practice because of its short analysis time, the need for a small amount of plasma, high specificity, and accuracy.

Measurement of piperacillin plasma concentrations in cancer patients with suspected infection.

BACKGROUND: Piperacillin (PIP) in combination with tazobactam is commonly used for anti-infective treatment in cancer patients. PIP exerts a time-dependent killing. Thus, the maintenance of plasma concentrations above a pre-defined target concentration for a pre-defined time may be relevant for optimal efficacy. It is assumed that PIP-plasma concentrations above the clinical breakpoint of the target pathogen [Pseudomonas aeruginosa, clinical breakpoint at minimal inhibitory concentration (MIC) 16 mg/L] should be reached for 100% of the dosing interval or >4xMIC (64 mg/L) for 50% of the dosing interval. Whereas studies in the intensive-care setting have shown underdosing in patients with sepsis, little is known about PIP-plasma concentrations in cancer patients. METHODS: Data of 56 cancer patients who received piperacillin/tazobactam (PIP/TAZ, 4.5 g three times daily) as empiric therapy for suspected infection were analysed at baseline and 4 h after the infusion. RESULTS: Median trough concentrations in steady state [median 3 days (IQR 3-5) after start of PIP/TAZ] were 4.6 mg/L (95% CI 0.3-136.3) and median PIP-plasma concentrations 4 h after infusion were 46.2 mg/L (95% CI 10.1-285.6). A second evaluation 5 days (IQR 4-7) after start of PIP/TAZ confirmed these results: trough concentrations were 2.7 mg/L (95% CI 0.5-6.3), concentrations after 4 h 28.0 mg/L (95% CI 1.7-47.3). A good renal function was associated with lower plasma concentrations (r = -0.388, p < 0.003). Detailed pharmacokinetic measurements in six patients showed low maximum plasma concentration (median 165 mg/L) and a rapid decline of plasma concentrations (median plasma half time 1.38 h). CONCLUSION: In conclusion, piperacillin plasma concentrations in cancer patients are below target levels warranting prospective trials to investigate therapeutic drug monitoring.

Histidine degradation via an aminotransferase increases the nutritional flexibility of Candida glabrata.

The ability to acquire nutrients during infections is an important attribute in microbial pathogenesis. Amino acids are a valuable source of nitrogen if they can be degraded by the infecting organism. In this work, we analyzed histidine utilization in the fungal pathogen of humans Candida glabrata. Hemiascomycete fungi, like C. glabrata or Saccharomyces cerevisiae, possess no gene coding for a histidine ammonia-lyase, which catalyzes the first step of a major histidine degradation pathway in most other organisms. We show that C. glabrata instead initializes histidine degradation via the aromatic amino acid aminotransferase Aro8. Although ARO8 is also present in S. cerevisiae and is induced by extracellular histidine, the yeast cannot use histidine as its sole nitrogen source, possibly due to growth inhibition by a downstream degradation product. Furthermore, C. glabrata relies only on Aro8 for phenylalanine and tryptophan utilization, since ARO8, but not its homologue ARO9, was transcriptionally activated in the presence of these amino acids. Accordingly, an ARO9 deletion had no effect on growth with aromatic amino acids. In contrast, in S. cerevisiae, ARO9 is strongly induced by tryptophan and is known to support growth on aromatic amino acids. Differences in the genomic structure of the ARO9 gene between C. glabrata and S. cerevisiae indicate a possible disruption in the regulatory upstream region. Thus, we show that, in contrast to S. cerevisiae, C. glabrata has adapted to use histidine as a sole source of nitrogen and that the aromatic amino acid aminotransferase Aro8, but not Aro9, is the enzyme required for this process.

Targeted metabolomics for discrimination of systemic inflammatory disorders in critically ill patients.

The occurrence of systemic inflammatory response syndrome (SIRS) remains a major problem in intensive care units with high morbidity and mortality. The differentiation between noninfectious and infectious etiologies of this disorder is challenging in routine clinical practice. Many biomarkers have been suggested for this purpose; however, sensitivity and specificity even of high-ranking biomarkers remain insufficient. Recently, metabolic profiling has attracted interest for biomarker discovery. The objective of this study was to identify metabolic biomarkers for differentiation of SIRS/sepsis. A total of 186 meta-bolites comprising six analyte classes were determined in 143 patients (74 SIRS, 69 sepsis) by LC-MS/MS. Two markers (C10:1 and PCaaC32:0) revealed significantly higher concentrations in sepsis. A classification model comprising these markers resulted in 80% and 70% correct classifications in a training set and a test set, respectively.This study demonstrates that acylcarnitines and glycerophosphatidylcholines may be helpful for differentiation of infectious from noninfectious systemic inflammation due to their significantly higher concentration in sepsis patients. Considering the well known pathophysiological relevance of lipid induction by bacterial components, metabolites as identified in this study are promising biomarker candidates in the differential diagnosis of SIRS and sepsis.

Minimal-invasive magnetic heating of tumors does not alter intra-tumoral nanoparticle accumulation, allowing for repeated therapy sessions: an in vivo study in mice.

Localized magnetic heating treatments (hyperthermia, thermal ablation) using superparamagnetic iron oxide nanoparticles (MNPs) continue to be an active area of cancer research. For generating the appropriate heat to sufficiently target cell destruction, adequate MNP concentrations need to be accumulated into tumors. Furthermore, the knowledge of MNP bio-distribution after application and additionally after heating is significant, firstly because of the possibility of repeated heating treatments if MNPs remain at the target region and secondly to study potential adverse effects dealing with MNP dilution from the target region over time. In this context, little is known about the behavior of MNPs after intra-tumoral application and magnetic heating. Therefore, the present in vivo study on the bio-distribution of intra-tumorally injected MNPs in mice focused on MNP long term monitoring of pre and post therapy over seven days using multi-channel magnetorelaxometry (MRX). Subsequently, single-channel MRX was adopted to study the bio-distribution of MNPs in internal organs and tumors of sacrificed animals. We found no distinct change of total MNP amounts in vivo during long term monitoring. Most of the MNP amounts remained in the tumors; only a few MNPs were detected in liver and spleen and less than 1% of totally injected MNPs were excreted. Apparently, the application of magnetic heating and the induction of apoptosis did not affect MNP accumulation. Our results indicate that MNP mainly remained within the injection side after magnetic heating over a seven-days-observation and therefore not affecting healthy tissue. As a consequence, localized magnetic heating therapy of tumors might be applied periodically for a better therapeutic outcome.

Characterization of iron oxide nanoparticles adsorbed with cisplatin for biomedical applications.

The aim of this study was to characterize the behaviour of cisplatin adsorbed magnetic nanoparticles (cis-MNPs) for minimal invasive cancer treatments in preliminary in vitro investigations. Cisplatin was adsorbed to magnetic nanoparticles (MNPs) by simple incubation. For stability determinations, cis-MNPs were incubated in dH(2)O, phosphate-buffered saline (PBS) and fetal calf serum (FCS) at 4-121 degrees C up to 20 weeks. Hydrodynamic diameters were measured using laser diffraction. The extent of cisplatin linkage was determined by atomic absorption spectrometry. The magnetite core size was assessed by vibrating sample magnetometry and transmission electron microscopy. The specific loss power (SLP) was measured in an alternating magnetic field. Our results showed that a maximum of 10.3 +/- 1.6 (dH(2)O), 10 +/- 1.6 (PBS) and 13.4 +/- 2.2 (FCS) mg cisplatin g(-1) Fe could be adsorbed to MNPs. With hyperthermal (42 degrees C) or thermal ablative (60 degrees C) temperatures, used for therapeutic approaches, cisplatin did not desorb from cis-MNPs in dH(2)O during incubation times of 180 or 30 min, respectively. In PBS and FCS, cisplatin amounts adsorbed to MNPs decreased rapidly to approximately 50% and 25% at these temperatures. This cisplatin release will be necessary for successful chemotherapeutic activity and should increase the therapeutic effect of magnetic heating treatment in medicinal applications. The hydrodynamic diameters of MNPs or cis-MNPs were around 70 nm and magnetization data showed superparamagnetic behaviour. The obtained mean core diameter was around 12 nm. The SLP of the sample was calculated to be 75.5 +/- 1.6 W g(-1). In conclusion, cis-MNPs exhibit advantageous features for a facilitated desorption of cisplatin in biological media and the heating potential is adequate for hyperthermic treatments. Therefore, even though further detailed investigations are still necessary, tentative use in local tumour therapies aiming at a specific chemotherapeutic release in combination with magnetic heating seems to be feasible in the long term.

Long-term prevalence of NIRF-labeled magnetic nanoparticles for the diagnostic and intraoperative imaging of inflammation.

Inflammation is a very common disease worldwide. In severe cases, surgery is often the method of choice. Today, there is a general need for the implementation of image-based guidance methodologies for reliable target resection. We investigated new near infrared fluorescence (NIRF)-nanoparticles (NPs) as a simple but effective bimodal magnetic resonance imaging (MRI) and optical contrast agent for diagnosis and intraoperative imaging of inflammation. Physicochemical analysis revealed that these NPs were highly fluorescent with similar characteristics like unlabeled NPs (hydrodynamic diameter about 130 nm and zeta potential about -10 mV). NP-uptake and NIR-dye labeling was biocompatible to macrophages (no impact on cellular ATP and reactive oxygen species production). These cells could successfully be tracked with MRI and NIRF-optical imaging. I.v. injection of fluorescent NPs into mice led to highly specific T2-weighted signal of edema due to uptake by phagocytic cells and subsequent migration to the site of inflammation. NIRF signals of the edema region were well detectable for up to 4 weeks, underlining the potential of the NPs for systematic planning and flexible time scheduling in intraoperative applications. NPs were degraded over a time period of 12 weeks, which was not altered due to inflammation. Redistribution of iron might be primarily due to inflammation and not to the presence of NPs per se in a concentration suitable for imaging. Our findings highlight the potential of the NPs to be used as a suitable tool for pre- and intraoperative imaging of inflammation.

Branched-chain amino acid catabolism is a conserved regulator of physiological ageing.

Ageing has been defined as a global decline in physiological function depending on both environmental and genetic factors. Here we identify gene transcripts that are similarly regulated during physiological ageing in nematodes, zebrafish and mice. We observe the strongest extension of lifespan when impairing expression of the branched-chain amino acid transferase-1 (bcat-1) gene in C. elegans, which leads to excessive levels of branched-chain amino acids (BCAAs). We further show that BCAAs reduce a LET-363/mTOR-dependent neuro-endocrine signal, which we identify as DAF-7/TGFß, and that impacts lifespan depending on its related receptors, DAF-1 and DAF-4, as well as ultimately on DAF-16/FoxO and HSF-1 in a cell-non-autonomous manner. The transcription factor HLH-15 controls and epistatically synergizes with BCAT-1 to modulate physiological ageing. Lastly and consistent with previous findings in rodents, nutritional supplementation of BCAAs extends nematodal lifespan. Taken together, BCAAs act as periphery-derived metabokines that induce a central neuro-endocrine response, culminating in extended healthspan.

Effect of thermomechanical pre-treatment on short- and long-term Ni release from biomedical NiTi.

The effect of annealing and deformation on short-term (21days) and long-term (8months) Ni release from biomedical NiTi wires is studied. The deformation of annealed NiTi wires causes cracking and flaking of the surface oxide layer. Flaking of oxide particles does not uncover the Ni-rich layer underneath the surface oxide layer, since at sites where flaking occurs, a thin (∼25nm) layer of oxide remains on top of this Ni-rich layer. The number of cracks in the oxide and Ni-rich layer, respectively, increases with deformation, and intercrystalline crack propagation into the Ni-rich layer and the NiTi bulk is observed. In plastically deformed wires, the cracks may remain opened, providing access of immersion liquid to these zones. Characteristics and quantity of short-term Ni release are significantly affected by the pre-deformation, resulting in an up to 2 times higher total Ni release within the first 21days of deformed compared to annealed wires. Pre-deformation does not significantly influence long-term Ni release; all annealed and deformed samples exhibit similar long-term Ni release rates. The source of Ni during short-term release is the Ni contained in the surface zone of the oxide layer. For high pre-deformation, the Ni-rich layer is a second source for Ni. This second source is also the cause for Ni release in long-term immersion experiments.

Evaluation of a straightforward and rapid method for the therapeutic drug monitoring of digitoxin by LC-MS/MS.

OBJECTIVES: Therapeutic drug monitoring of digitoxin is strongly recommended but metabolites of digitoxin and digitoxin-like immunoreactive substances may interfere with widely used immunoassays. Recently evaluated assays on LC-MS/MS have the drawback of long turnaround time. We sought to evaluate a specific method on LC-MS/MS optimizing sample preparation thereby significantly reducing turnaround time. DESIGN AND METHODS: Linearity, functional sensitivity, and precision of the method were established. External quality control samples were used for the evaluation of accuracy of the LS-MS/MS method. In addition, digitoxin concentrations in 221 samples were measured by LC-MS/MS and immunoassay. RESULTS: Linearity was validated between 0.15 and 80 ng/mL. Limit of quantification was established at 0.14 ng/mL. Between-day imprecision lay between 1.4 and 4.9% and meets the conditions required for routine analysis. Comparison to results obtained by immunoassay revealed a mean difference of -1.2 ng/mL. CONCLUSIONS: By optimizing preparation steps turnaround time was shorter for LC-MS/MS than for immunoassay. This did not result in increased susceptibility to matrix effects. Analytical performance was sufficient for routine analysis. Therefore, the method is suitable for routine therapeutic drug monitoring of digitoxin.

Can we accurately quantify nanoparticle associated proteins when constructing high-affinity MRI molecular imaging probes?

Targeted magnetic resonance contrast agents (e.g. iron oxide nanoparticles) have the potential to become highly selective imaging tools. In this context, quantification of the coupled amount of protein is essential for the design of antibody- or antibody fragment-conjugated nanoparticles. Nevertheless, the presence of magnetic iron oxide nanoparticles is still an unsolved problem for this task. The aim of the present work was to clarify whether proteins can be reliably quantified directly in the presence of magnetic iron oxide nanoparticles without the use of fluorescence or radioactivity. Protein quantification via Bradford was not influenced by the presence of magnetic iron oxide nanoparticles (0-17.2 mmol Fe l(-1) ). Instead, bicinchoninic acid based assay was, indeed, distinctly affected by the presence of nanoparticle-iron in suspension (0.1-17.2 mmol Fe l(-1) ), although the influence was linear. This observation allowed for adequate mathematical corrections with known iron content of a given nanoparticle. The applicability of our approach was demonstrated by the determination of bovine serum albumin (BSA) content coupled to dextrane-coated magnetic nanoparticles, which was found with the QuantiPro Bicinchoninic acid assay to be of 1.5 ± 0.2 µg BSA per 1 mg nanoparticle. Both Bradford and bicinchoninic acid assay protein assays allow for direct quantification of proteins in the presence of iron oxide containing magnetic nanoparticles, without the need for the introduction of radioactivity or fluorescence modules. Thus in future it should be possible to make more precise estimations about the coupled protein amount in high-affinity targeted MRI probes for the identification of specific molecules in living organisms, an aspect which is lacking in corresponding works published so far. Additionally, the present protein coupling procedures can be drastically improved by our proposed protein quantification method.