A semi-automated, isotope-dilution high-resolution mass spectrometry assay for therapeutic drug monitoring of antidepressants.

BACKGROUND: Therapeutic drug monitoring (TDM) of antidepressants is important to ensure compliance and to rule out pharmacokinetic abnormalities. Therefore, reliable methods for quantification are important for clinical laboratories. Most of the currently used mass spectrometry methods use triple quadrupoles as mass analyzers. We aimed to develop a method using high-resolution mass spectrometry (HRMS) and wanted to test the suitability of this analyzer for quantitative TDM assays. This would be beneficial since HRMS instruments can also be used for metabolomics and protein analysis and, thus, many different analyses could be run on one instrument. METHODS: After manual protein precipitation of serum samples, further sample clean-up was achieved using a Turbo Flow column preconnected to the analytical LC column. Stable isotope-labelled counterparts of the target analytes were used as internal standards. For detection, we used a Q Exactive Focus Orbitrap mass spectrometer operating in full-scan mode. Ionization was performed in positive ESI. RESULTS: Accuracy, recovery, and matrix effect were acceptable for all analytes. The method demonstrated outstanding precision (within-run imprecision <4.5%, between-run imprecision <7.5%). The selectivity of the method was ensured by chromatographic separation of all isobaric compounds. Close agreement between Orbitrap and triple stage based quantification was observed in a comparison measurement of leftover patient samples. CONCLUSIONS: We have established a selective method for the quantification of antidepressants with outstanding precision using a high-resolution Orbitrap mass spectrometer. The applicability of HRMS instruments to TDM was demonstrated.

The dynamics of a serum steroid profile after stimulation with intravenous ACTH.

BACKGROUND: Stimulation with intravenous adrenocorticotropic hormone (ACTH) is a widely used diagnostic procedure to characterize the adrenocortical function. Currently, the response of serum cortisol, mainly quantified by immunoassays, is the only established read-out of this test. By using liquid chromatography coupled with mass spectrometry (LC-MS/MS) simultaneous determination of several steroids that respond to ACTH stimulation is now possible. The aim of this study was to further characterize the typical effect of exogenous ACTH (250 mg) on a LC-MS/MS-serum steroid profile. METHODS: A set of 36 paired samples (pre-/post-IV-ACTH) was investigated (age range 22-58, 26 female and 10 male individuals). Serum steroid profiling was performed using a LC-MS/MS method covering cortisol, cortisone, corticosterone, 11-deoxycortisol, 17-OH-progesterone and 11-deoxycorticosterone. RESULTS: The concentrations of all measured steroids increased after stimulation with ACTH, except for cortisone. Serum corticosterone, 11-deoxycorticosterone and 11-deoxycortisol showed markedly more pronounced relative increases compared to cortisol. The strongest response was observed for corticosterone (15-fold median relative increase, compared to 1.4-fold median increase of cortisol). CONCLUSION: Serum steroid profiling using LC-MS/MS after stimulation with IV ACTH demonstrates highly dynamic response patterns. Further studies should address in particular serum corticosterone as a potential novel marker of biochemical stress response.

Biphenyl based stationary phases for improved selectivity in complex steroid assays.

The measurement of steroid hormones and their corticoid precursors is an important aspect in endocrinology since these analytes are biomarkers for several endocrine disorders. Over the last few years, HPLC-MS/MS has become the method of choice to analyze these compounds. There are already several methods using stationary phases modified with C18 groups. However, since these columns sometimes do not enable sufficient separation of some isobaric steroids, we investigated the potential of a different RP modification using biphenyl groups for the separation of challenging isobars such as corticosterone, 11- and 21-deoxycortisol. The aim of our work was the development of an isotope dilution UHPLC-MS/MS assay for clinical research that combines simple and effective sample preparation with a powerful MS method quantifying a broad steroid panel (aldosterone, corticosterone, cortisol, cortisone, 11-deoxycorticosterone, 11-deoxycortisol, 21-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, 17-OH-progesterone, progesterone, and testosterone) in human serum. After a manual protein precipitation step using zinc trifluoroacetate (ZnTFA) in methanol, the supernatants were directly injected into the UHPLC-MS system. Chromatographic baseline separation of all isobaric compounds (corticosterone↔11-deoxycortisol↔21-deoxycortisol, 17-OH-progesterone↔11-deoxycorticosterone, and aldosterone↔cortisone) was achieved using a Kinetex Biphenyl column (150×2.1mm, 1.7µm) with a mobile phase consisting of 0.2mM ammonium fluoride in water and methanol. The total run time was 10min. For detection we used a Xevo TQ-S mass spectrometer operating in the ESI positive and negative modes. The method was validated according to the EMA guideline for bioanalytical method validation. The results for accuracy (within-run: 92.3%-115%, between-run: 92.4 %-113%) and imprecision (within-run: 0.80%-9.05%, between-run: 1.98 %-15.2%) were satisfying. The recovery ranged from 95% to 111%. The matrix effect was between 93% and 112% and an excellent linearity with R2>0.99 for all analytes was achieved. It was demonstrated that biphenyl based columns are a powerful tool for comprehensive, MS based steroid assays including various isobaric substances. Additionally, we could evince that ZnTFA is a convenient precipitation agent suitable for steroid analysis.

An LC-MS/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material.

BACKGROUND: Accurate measurement of gentamicin concentration in serum and plasma is required for therapeutic drug monitoring to ensure appropriate treatment of patients. In this work, we present a validated LC-MS/MS-based candidate reference measurement procedure for total gentamicin quantification to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring of this compound. Total gentamicin is the sum of the concentrations of five known congeners C1, C1a, C2, C2a and C2b. To our knowledge, there is so far no LC-MS method for quantification of total gentamicin in human serum described in literature. METHODS: Sample preparation was based on sample dilution with an aqueous internal standard solution followed by protein precipitation. Stable derivatives of gentamicin-glycine congeners were prepared by chemical synthesis and used as internal standards. The primary calibration material used in this assay was characterized by NMR spectroscopy and the pattern of the gentamicin congeners was determined. The total gentamicin was reported as the sum of the congeners which were quantified individually by LC-MS/MS. RESULTS: The method allows the measurement of total gentamicin in human serum and plasma in the concentration range of 0.1 to 12.0µg/ml with an assay imprecision of ≤6% CV and an assay accuracy between 96% and 114%. LOD and LOQ for the total gentamicin were 0.04µg/ml and 0.13µg/ml, respectively. Comparative measurement of 128 native patient samples using this method implemented at two laboratory sites showed an excellent agreement. CONCLUSIONS: Validation results proved that this protocol describes a robust and reliable method which is suggested as reference measurement procedure for the standardization and harmonization of routine assays for the quantification of total gentamicin.

Ferromagnetic particles as a rapid and robust sample preparation for the absolute quantification of seven eicosanoids in human plasma by UHPLC-MS/MS.

We used ferromagnetic particles as a novel technique to deproteinize plasma samples prior to quantitative UHPLC-MS/MS analysis of seven eicosanoids [thromboxane B2 (TXB2), prostaglandin E2 (PGE2), PGD2, 5-hydroxyeicosatetraenoic acid (5-HETE), 11-HETE, 12-HETE, arachidonic acid (AA)]. A combination of ferromagnetic particle enhanced deproteination and subsequent on-line solid phase extraction (on-line SPE) realized quick and convenient semi-automated sample preparation-in contrast to widely used manual SPE techniques which are rather laborious and therefore impede the investigation of AA metabolism in larger patient cohorts. Method evaluation was performed according to a protocol based on the EMA guideline for bioanalytical method validation, modified for endogenous compounds. Calibrators were prepared in ethanol. The calibration curves were found to be linear in a range of 0.1-80ngmL(-1) (TXB2, PGE2, PGD2), 0.05-40ngmL(-1) (5-HETE, 11-HETE), 0.5-400ngmL(-1) (12-HETE) and 25-9800ngmL(-1) (AA). Regarding all analytes and all quality controls, the resulting precision data (inter-assay 2.6 %-15.5 %; intra-assay 2.5 %-15.1 %, expressed as variation coefficient) as well as the accuracy results (inter-assay 93.3 %-125 %; intra-assay 91.7 %-114 %) were adequate. Further experiments addressing matrix effect, recovery and robustness, yielded also very satisfying results. As a proof of principle, the newly developed LC-MS/MS assay was employed to determine the capacity of AA metabolite release after whole blood stimulation in healthy blood donors. For this purpose, whole blood specimens of 5 healthy blood donors were analyzed at baseline and after a lipopolysaccharide (LPS) induced blood cell activation. In several baseline samples some eicosanoids levels were below the Lower Limit of Quantification. However, in the stimulated samples all chosen eicosanoids (except PGD2) could be quantified. These results, in context with those obtained in validation, demonstrate the applicability of ferromagnetic particles for the sample preparation for eicosanoids in human plasma. Thus, we conclude that ferromagnetic particle enhanced deproteination is a promising novel tool for sample preparation in LC-MS/MS, which is of particular interest for automation in clinical mass spectrometry, e.g. in order to further address eicosanoid analysis in larger patient cohorts.

Isotope Inversion Experiment evaluating the suitability of calibration in surrogate matrix for quantification via LC-MS/MS-Exemplary application for a steroid multi-method.

For quotable quantitative analysis of endogenous analytes in complex biological samples by isotope dilution LC-MS/MS, the creation of appropriate calibrators is a challenge, since analyte-free authentic material is in general not available. Thus, surrogate matrices are often used to prepare calibrators and controls. However, currently employed validation protocols do not include specific experiments to verify the suitability of a surrogate matrix calibration for quantification of authentic matrix samples. The aim of the study was the development of a novel validation experiment to test whether surrogate matrix based calibrators enable correct quantification of authentic matrix samples. The key element of the novel validation experiment is the inversion of nonlabelled analytes and their stable isotope labelled (SIL) counterparts in respect to their functions, i.e. SIL compound is the analyte and nonlabelled substance is employed as internal standard. As a consequence, both surrogate and authentic matrix are analyte-free regarding SIL analytes, which allows a comparison of both matrices. We called this approach Isotope Inversion Experiment. As figure of merit we defined the accuracy of inverse quality controls in authentic matrix quantified by means of a surrogate matrix calibration curve. As a proof-of-concept application a LC-MS/MS assay addressing six corticosteroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycorticosterone, and 17-OH-progesterone) was chosen. The integration of the Isotope Inversion Experiment in the validation protocol for the steroid assay was successfully realized. The accuracy results of the inverse quality controls were all in all very satisfying. As a consequence the suitability of a surrogate matrix calibration for quantification of the targeted steroids in human serum as authentic matrix could be successfully demonstrated. The Isotope Inversion Experiment fills a gap in the validation process for LC-MS/MS assays quantifying endogenous analytes. We consider it a valuable and convenient tool to evaluate the correct quantification of authentic matrix samples based on a calibration curve in surrogate matrix.

MS Binding Assays for the Three Monoamine Transporters Using the Triple Reuptake Inhibitor (1R,3S)-Indatraline as Native Marker.

We herein present label-free, mass-spectrometry-based binding assays (MS Binding Assays) for the human dopamine, norepinephrine, and serotonin transporters (hDAT, hNET, and hSERT). Using this approach both enantiomers of the triple reuptake inhibitor indatraline as well as its cis-configured diastereomer were investigated toward hDAT, hNET, and hSERT in saturation experiments. The dissociation rate constants for (1R,3S)-indatraline binding at hDAT, hNET, and hSERT were determined in kinetic studies. These experiments revealed an allosteric effect of clomipramine on the dissociation of (1R,3S)-indatraline from hSERT. Finally, a comprehensive set of known monoamine transport inhibitors and substrates was studied in competition experiments at hDAT, hNET, and hSERT, using (1R,3S)-indatraline as nonlabeled marker. The results are in excellent agreement with those reported for radioligand binding assays. Therefore, the established MS Binding Assays are a promising alternative to the latter for the characterization of new monoamine reuptake inhibitors at DAT, NET, and SERT.

Development and validation of an LC-ESI-MS/MS method for the triple reuptake inhibitor indatraline enabling its quantification in MS Binding Assays.

We herein present the first LC-MS/MS quantification method for indatraline, a highly potent nonselective inhibitor of the three monoamine transporters (for dopamine, DAT; norepinephrine, NET; serotonin, SERT), and its application to MS Binding Assays. For HPLC, an R18 column with a mobile phase composed of acetonitrile and ammonium bicarbonate buffer (5 mmol L(-1), pH 10.0) in a ratio of 90:10 (v/v) at a flow rate of 600 µL min(-1) was used. Recording indatraline at m/z 292.2/261.0 and ((2)H(7))-indatraline, employed as internal standard, at m/z 299.2/268.0 allowed reliable quantification from 5 pmol L(-1) (LLOQ) to 5 nmol L(-1) in biological matrices without additional sample preparation. Validation of the developed quantification method showed that selectivity, calibration standard curve, accuracy, as well as precision meet the criteria of the CDER guideline. Applying this method to mass spectrometry (MS) Binding Assays, a label-free MS-based alternative to conventional radioligand binding assays, binding of indatraline's eutomer, (1R,3S)-indatraline, towards NET could be characterized directly for the first time, revealing an equilibrium dissociation constant (K d) of 805 pmol L(-1). Additionally, it could be shown that the established MS Binding Assays enable characterization of test compounds in competition experiments. As the established setup is based on a 96-well format and an LC MS/MS method with a short chromatographic cycle time (1.5 min), the developed MS Binding Assays enable considerable throughput and are therefore well suited as substitute for corresponding radioligand binding assays.

Protein damage, repair and proteolysis.

Proteins are continuously affected by various intrinsic and extrinsic factors. Damaged proteins influence several intracellular pathways and result in different disorders and diseases. Aggregation of damaged proteins depends on the balance between their generation and their reversal or elimination by protein repair systems and degradation, respectively. With regard to protein repair, only few repair mechanisms have been evidenced including the reduction of methionine sulfoxide residues by the methionine sulfoxide reductases, the conversion of isoaspartyl residues to L-aspartate by L-isoaspartate methyl transferase and deglycation by phosphorylation of protein-bound fructosamine by fructosamine-3-kinase. Protein degradation is orchestrated by two major proteolytic systems, namely the lysosome and the proteasome. Alteration of the function for both systems has been involved in all aspects of cellular metabolic networks linked to either normal or pathological processes. Given the importance of protein repair and degradation, great effort has recently been made regarding the modulation of these systems in various physiological conditions such as aging, as well as in diseases. Genetic modulation has produced promising results in the area of protein repair enzymes but there are not yet any identified potent inhibitors, and, to our knowledge, only one activating compound has been reported so far. In contrast, different drugs as well as natural compounds that interfere with proteolysis have been identified and/or developed resulting in homeostatic maintenance and/or the delay of disease progression.

Enantiopurity determination of the enantiomers of the triple reuptake inhibitor indatraline.

The present study describes the development of two approaches for the determination of the enantiopurity of both enantiomers of indatraline. Initially, a method was developed using different chiral solvating agents (CSAs) for diastereomeric discrimination regarding signal separation in (1)H nuclear magnetic resonance (NMR) spectroscopy, revealing MTPA as a promising choice for the differentiation of the indatraline enantiomers. This CSA was also tested for its ideal molar ratio, temperature, and solvent. Optimized conditions could be achieved that made determination of enantiopurity for (1R,3S)-indatraline up to 98.9% enantiomeric excess (ee) possible. To quantify even higher enantiopurities, a high-performance liquid chromatography (HPLC) method based on a modified ß-cyclodextrine phase was established. The influence of buffer type, concentration, pH value, percentage and kind of organic modifier, temperature, injection volume as well as sample solvent on chromatographic parameters was investigated. Afterwards, the reliability of the established HPLC method was demonstrated by validation according to the ICH guideline Q2(R1) regarding specificity, accuracy, precision, linearity, and quantitation limit. The developed method proved to be strictly linear within a concentration range of 1.25-1000 µM for the (1R,3S)-enantiomer and 1.25-750 µM for its mirror image that enables a reliable determination of enantiopurities up to 99.75% ee for the (1R,3S)-enantiomer and up to 99.67% ee for the (1S,3R)-enantiomer.

Diet-derived advanced glycation end products or lipofuscin disrupts proteostasis and reduces life span in Drosophila melanogaster.

Advanced glycation end product (AGE)-modified proteins are formed by the nonenzymatic glycation of free amino groups of proteins and, along with lipofuscin (a highly oxidized aggregate of covalently cross-linked proteins, sugars, and lipids), have been found to accumulate during aging and in several age-related diseases. As the in vivo effects of diet-derived AGEs or lipofuscin remain elusive, we sought to study the impact of oral administration of glucose-, fructose-, or ribose-modified albumin or of artificial lipofuscin in a genetically tractable model organism. We report herein that continuous feeding of young Drosophila flies with culture medium enriched in AGEs or in lipofuscin resulted in reduced locomotor performance and in accelerated rates of AGE-modified proteins and carbonylated proteins accumulation in the somatic tissues and hemolymph of flies, as well as in a significant reduction of flies health span and life span. These phenotypic effects were accompanied by reduced proteasome peptidase activities in both the hemolymph and the somatic tissues of flies and higher levels of oxidative stress; furthermore, oral administration of AGEs or lipofuscin in flies triggered an upregulation of the lysosomal cathepsin B, L activities. Finally, RNAi-mediated cathepsin D knockdown reduced flies longevity and significantly augmented the deleterious effects of AGEs and lipofuscin, indicating that lysosomal cathepsins reduce the toxicity of diet-derived AGEs or lipofuscin. Our in vivo studies demonstrate that chronic ingestion of AGEs or lipofuscin disrupts proteostasis and accelerates the functional decline that occurs with normal aging.

Biomarkers of oxidative stress, antioxidant defence and inflammation are altered in the senescence-accelerated mouse prone 8.

In this study we compared biomarkers of oxidative stress, stress response, antioxidant defence and inflammation between mice (n = 10 per group, female, 7 months old) with an accelerated (SAMP8) and a normal ageing phenotype (SAMR1). As compared to SAMR1 mice, SAMP8 mice exhibited higher levels of lipid peroxides and protein carbonyls as well as a lower activity of the proteasomal subunit ß-5. Furthermore, heme oxygenase-1 and paraoxonase-1 (PON-1) status was lower in SAMP8 mice indicating impaired stress response. Biomarkers of inflammation such as C-reactive protein and serum amyloid P were elevated in SAMP8 mice. Interestingly, impaired stress response and increased inflammation in SAMP8 mice were associated with elevated concentrations of ascorbic acid and α-tocopherol in the liver. An age-dependent increase in hepatic vitamin E and a decline in PON-1 gene expression were also observed in aged compared to young C57BL/6 mice.

Lipofuscin is formed independently of macroautophagy and lysosomal activity in stress-induced prematurely senescent human fibroblasts.

In the current literature, the lysosomal system is considered to be involved in the intracellular formation and accumulation of lipofuscin, a highly oxidized and covalently cross-linked aggregate of proteins that fills the lysosomal volume during aging. In contrast, our experimental results presented here suggest that both the autophagosomes and the lysosomal system are not mandatory for the formation of lipofuscin, since that material accumulates in the cytosolic volume if autophagy or lysosomal activity is inhibited. However, such an inhibition is accompanied by an enhanced toxicity of the formed protein aggregates. Furthermore, it could be proven that macroautophagy is responsible for the uptake of lipofuscin into the lysosomes.

Advanced-glycation-end-product-induced formation of immunoproteasomes: involvement of RAGE and Jak2/STAT1.

AGEs (advanced glycation-end products) accumulate during aging and several pathologies such as Alzheimer's disease and diabetes. These protein products are known to inhibit proteolytic pathways. Moreover, AGEs are known to be involved in the activation of immune responses. In the present study we demonstrate that AGEs induce the expression of immunoproteasomal subunits. To elucidate a molecular basis underlying the observed effects we were able to demonstrate an activation of the Jak2 (Janus kinase 2)/STAT1 (signal transducer and activator of transcription 1) pathway. Inhibition of Jak2 by AG-490 and STAT1 by specific siRNA (small interfering RNA) abolished AGE-induced expression of immunoproteasomal subunits. Furthermore, silencing of RAGE (receptor for AGEs) revealed that AGE-induced up-regulation of the immunoproteasome is mediated by a RAGE signalling process. Thus we have described for the first time that the signalling pathway of Jak2 and STAT1 activated by AGEs via RAGE is involved in the induction of the immunoproteasome.

Rapid and sensitive determination of protein-nitrotyrosine by ELISA: Application to human plasma.

3-Nitrotyrosine (3NT) is known as an important indicator of nitrosative stress and has been linked to various diseases. Our aim was to develop an indirect ELISA (enzyme-linked immunosorbent assay) method suitable for the detection of protein-bound 3NT in clinical plasma and serum samples. Nitrated protein standards and reduced protein standards were prepared. Limit of detection was determined for standards; recovery and reproducibility were determined for human plasma samples. The limit of detection for this method is 1.82±0.56 pmol/mg protein. Mean recovery of standards was 95%. 3NT concentration in plasma samples of obese and normal weight subjects was determined to be between 2 pmol/mg and 19 pmol/mg. No time-consuming sample preparation or expensive laboratory equipment is required, and applied antibodies are commercially available. Sensitivity, rapid analysis time, possibilities of high throughput applications and small sample volumes make this ELISA attractive for use in clinical laboratories.

A diet rich in olive oil phenolics reduces oxidative stress in the heart of SAMP8 mice by induction of Nrf2-dependent gene expression.

A Mediterranean diet rich in olive oil has been associated with health benefits in humans. It is unclear if and to what extent olive oil phenolics may mediate these health benefits. In this study, we fed senescence-accelerated mouse-prone 8 (SAMP8, n=11 per group) semisynthetic diets with 10% olive oil containing either high (HP) or low amounts of olive oil phenolics (LP) for 4.5 months. Mice consuming the HP diet had significantly lower concentrations of the oxidative damage markers thiobarbituric acid-reactive substances and protein carbonyls in the heart, whereas proteasomal activity was similar in both groups. Nrf2-dependent gene expression may be impaired during the aging process. Therefore, we measured Nrf2 and its target genes glutathione-S-transferase (GST), γ-glutamyl cysteine synthetase (γ-GCS), nicotinamide adenine dinucleotide phosphate [NAD(P)H]:quinone oxidoreductase (NQO1), and paraoxonase-2 (PON2) in the hearts of these mice. Nrf2 as well as GST, γ-GCS, NQO1, and PON2 mRNA levels were significantly higher in heart tissue of the HP as compared to the LP group. The HP-fed mice had significantly higher PON1 activity in serum compared to those receiving the LP diet. Furthermore, HP feeding increased relative SIRT1 mRNA levels. Additional mechanistic cell culture experiments were performed, and they suggest that the olive oil phenolic hydroxytyrosol present in the HP oil may be responsible for the induction of Nrf2-dependent gene expression and the increase in PON activity. In conclusion, a diet rich in olive oil phenolics may prevent oxidative stress in the heart of SAMP8 mice by modulating Nrf2-dependent gene expression.

Cathepsins D and L reduce the toxicity of advanced glycation end products.

Advanced glycation end product-modified proteins are known for accumulating during aging and in several pathological conditions such as diabetes, renal failure, and neurodegenerative disorders. There is little information about the intracellular fate of endocytosed advanced glycation end products (AGEs) and their influence on proteolytic systems. However, it is known that the lysosomal system is impaired during aging. Therefore, undegraded material may accumulate and play a considerable role in the development of diverse diseases. To investigate if AGEs can be degraded and to test whether they accumulate because of impaired lysosomal proteases we studied the effects of advanced glycation end products on the endosomal-lysosomal system. Five different types of AGEs were generated by bovine serum albumin incubation with glyoxal, methylglyoxal, glucose, fructose, and ribose. The first experiments revealed the uptake of AGEs by the macrophage cell line RAW 264.7. Further investigations demonstrated an increase in cathepsin D and L activity and an increase in mature cathepsins D and L. Increased activities were accompanied by the presence of more lysosomes, measured by staining with LysoTracker blue. To specify the roles of cathepsins D and L we used knockout cells to test the roles of both cathepsins on the toxicity of advanced glycation end products. In summary we conclude that both cathepsins are required for a reduction in advanced glycation end product-induced cytotoxicity.

The outcome of 5-ALA-mediated photodynamic treatment in melanoma cells is influenced by vitamin C and heme oxygenase-1.

Photodynamic therapy (PDT) is an important clinical approach for cancer treatment. It involves the administration of a photosensitizer, followed by its activation with light and induction of cell death. The underlying mechanism is an increased production of reactive oxygen species (ROS) leading to oxidative stress, which is followed by cell death. However, effectiveness of PDT is limited due to an initiation of endogenous rescue response systems like heme oxygenase-1 (HO-1) in tumor cells. In recent years, consuming of antioxidant supplements has become widespread, but the effect of exogenously applied antioxidants on cancer therapy outcome remains unclear. Thus, this study was aimed to investigate if exogenous antioxidants might decrease ROS-induced cytotoxicity in photodynamic treatment. Lycopene, ß-carotene, vitamin C, N-acetylcysteine, trolox, and N-tert-butyl-α-phenylnitrone in different doses were administered to human melanoma cells prior exposure to photodynamic treatment. Supplementation with vitamin C resulted in a significant decrease of the cell death rate, whereas the other tested antioxidants had no effect on cell viability and oxidative stress markers. The simultaneous application of vitamin C with the HO-1 activity inhibitor zinc protoporphyrine IX (ZnPPIX) caused a considerable decrease of photodynamic treatment-induced cytotoxicity compared to ZnPPIX alone. It can be summarized that exogenously applied antioxidants do not have a leading role in the protective response against photodynamic treatment. However, further studies are necessary to investigate more antioxidants and other substances, which might affect the outcome of photodynamic treatment in cancer therapy.

Protein oxidative modifications in the ageing brain: consequence for the onset of neurodegenerative disease.

The free radical theory of ageing proposes the accumulation of altered, less active and toxic molecules of DNA, RNA, proteins and lipids caused by reactive oxygen species and reactive nitrogen species. Neurodegenerative disorders are characterized by an abnormal accumulation of oxidatively damaged macromolecules inside cells and in the extracellular space. Proteins involved in the formation of aggregates are ß-amyloid, tau, α-synuclein, parkin, prion proteins and proteins containing polyglutamine. These abnormal aggregated proteins influence normal cellular metabolism. Additionally, deposition of abnormal proteins induces oxidative stress and proteasomal as well as mitochondrial dysfunction that ultimately lead to neuronal cell death. This review focuses on the impact of oxidative and nitrative stress in the ageing brain and, consequently, on the generation of modified proteins, as these post-translational modifications are assumed to play an important role in the development of neurodegenerative diseases.

Diagnostic yield of ink-jet prints from digital radiographs for the assessment of approximal carious lesions: ROC-analysis.

AIMS: To investigate the diagnostic quality of different quality, individually calibrated ink-jet printers for the very challenging dental radiographic task of approximal carious lesion detection. MATERIALS AND METHODS: A test-pattern evaluating resolution, contrast and homogeneity of the ink-jet prints was developed. 50 standardized dental radiographs each showing two neighbouring teeth in natural contact were printed on glossy paper with calibrated, randomly selected ink-jet printers (Canon S520 and iP4500, Epson Stylus Photo R2400). Printing size equalled the viewing size on a 17″ cathode-ray-tube monitor daily quality-tested according to German regulations. The true caries status was determined from serial sectioning and microscopic evaluation. 16 experienced observers evaluated the radiographs on a five-point confidence scale on all prints plus the viewing monitor with respect to the visibility of a carious lesion. A non-parametric Receiver-Operating Characteristics (ROC-) analysis was performed explicitly designed for the evaluation of readings stemming from identical samples but different modality. Significant differences are expressed by a critical ratio z exceeding ±2. Diagnostic accuracy was determined by the area (Az) underneath the ROC-curves. RESULTS: Average Az-values ranged between 0.62 (S520 and R2400) and 0.64 (monitor, iP4500), with no significant difference between modalities (P=0.172). Neither significant (range mean z: -0.40 (S520) and -0.11 (iP4500)) nor clinically relevant differences were found between printers and viewing monitor. CONCLUSIONS: Our results for a challenging task in dental radiography indicate that calibrated, off-the-shelf ink-jet printers are able to reproduce (dental) radiographs at quality levels sufficient for radiographic diagnosis in a typical dental working environment.