Alpha synuclein dimers and oligomers are increased in overexpressing conditions in vitro and in vivo.

Parkinson's disease is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta along with the formation of intracellular fibrillar inclusions (Lewy bodies and Lewy neuritis), which are mainly composed of aggregated α-synuclein (ASYN). This latter is a 14 kDa protein that localizes to synaptic vesicles in nerve terminals and promotes soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex assembly. We explored the monomeric and oligomeric state of ASYN in vitro in HEK293s and SH-SY5Y cell lines. In addition rats were injected in the substantia nigra with an Adeno associated virus carrying the human A53T mutation of ASYN (in vivo experiments). We show that human wild type ASYN as well as PD-linked mutations (A30P, E46K and A53T) in overexpressing conditions mostly exists in a monomeric state in equilibrium with dimeric forms. The monomer/dimer ratio is unaffected by PD-linked mutation. Furthermore, the A30P, E46K and A53T mutations overexpression strongly increased cell death compared to wild type ASYN. Taken together, our data suggest that ASYN dimers amount do not directly correlate to reduced cellular viability, suggesting a different role in protein function and induced pathology. Our data suggest that early ASYN neuro-pathogenic effects are probably mediated by other molecular processes than increased oligomerization alone.

Continuous drug delivery in early- and late-stage Parkinson's disease as a strategy for avoiding dyskinesia induction and expression.

The treatment of the motor symptoms of Parkinson's disease (PD) is dependent on the use of dopamine replacement therapy in the form of L: -dopa and dopamine agonist drugs. However, the development of dyskinesia (chorea, dystonia, athetosis) can become treatment limiting. The initiation of dyskinesia involves a priming process dependent on the presence of nigral dopaminergic cell loss leading to alterations in basal ganglia function that underlie the expression of involuntary movements following the administration of each drug dose. Once established, dyskinesia is difficult to control and it is even more difficult to reverse the priming process. Dyskinesia is more commonly induced by L: -dopa than by dopamine agonist drugs. This has been associated with the short duration of L: -dopa causing pulsatile stimulation of postsynaptic dopamine receptors compared to the longer acting dopamine agonists that cause more continuous stimulation. As a result, the concept of continuous dopaminergic stimulation (CDS) has arisen and has come to dominate the strategy for treatment of early PD. However, CDS has flaws that have led to the general acceptance that continuous drug delivery (CDD) is key to the successful treatment of PD. Studies in both experimental models of PD and in clinical trials have shown CDD to improve efficacy, but reduce dyskinesia induction, and to reverse established involuntary movements. Two key clinical strategies currently address the concept of CDD: (1) in early-, mid- and late-stage PD, transdermal administration of rotigotine provides 24 h of drug delivery; (2) in late-stage PD, the constant intraduodenal administration of L: -dopa is utilized to improve control of motor symptoms and to diminish established dyskinesia. This review examines the rationale for CDD and explores the clinical benefit of using such a strategy for the treatment of patients with PD.

Transcriptional alterations under continuous or pulsatile dopaminergic treatment in dyskinetic rats.

Continuous dopaminergic treatment is considered to prevent or delay the occurrence of dyskinesia in patients with Parkinson's disease (PD). Rotigotine is a non-ergolinic D(3) > D(2) > D(1) dopamine-receptor agonist for the treatment of PD using a transdermal delivery system providing stable plasma levels. We aimed to investigate the differential influence on gene expression of pulsatile L: -DOPA or rotigotine versus a continuous rotigotine treatment. The gene expression profile within the nigro-striatal system of unilateral 6-hydroxydopamine-lesioned rats was assessed in order to differentiate potential changes in gene expression following the various treatment using Affymetrix microarrays and quantitative RT-PCR. The expression of 15 genes in the substantia nigra and of 11 genes in the striatum was altered under pulsatile treatments inducing dyskinetic motor response, but was unchanged under continuous rotigotine treatment that did not cause dyskinetic motor response. The route of administration of a dopaminergic drug is important for the induction or prevention of motor abnormalities and adaptive gene expressions. The decline of neurotrophin-3 expression under pulsatile administration was considered of particular importance.

Rotigotine transdermal patch for the treatment of Parkinson's disease and restless legs syndrome.

The nonergoline dopamine agonist rotigotine, is delivered transdermally using a silicone-based patch (Neupro(R); UCB Pharma GmbH), which promotes unidirectional drug flow from the transdermal system to the skin. Pharmacokinetic data show stable steady-state plasma concentrations over 24 h, maintained with once-daily patch administration. Stable plasma concentrations are reflected by stable concentrations in the brain, as has been shown in animal studies. This suggests a continuous stimulation of dopaminergic receptors, which may result in a reduction in or prevention of abnormal involuntary movements in Parkinson's disease (PD) after prolonged treatment. Clinical trials have demonstrated that rotigotine is efficacious as monotherapy for PD and restless legs syndrome (RLS), and open-label extension studies have shown its long-term efficacy. Furthermore, rotigotine can be used effectively in coadministration with levodopa, enabling a reduction of the levodopa treatment doses in PD. Transdermal application also yields favorable pharmacokinetics for rotigotine: rapid metabolism and lack of skin accumulation allow for good control of chronic administration or withdrawal by patch removal, and the transdermal application approach circumvents problems of gastrointestinal absorption and enables administration prior to, during or following surgery. In addition, no dose adaptation is required regarding gender or ethnicity, or for patients with impaired liver or kidney function or on hemodialysis. The safety profile of rotigotine transdermal patch is favorable; common side effects attributed to transdermal delivery or dopaminergic stimulation are generally mild to moderate in intensity. Importantly, augmentation of RLS is uncommon under long-term rotigotine treatment and dyskinesia in PD patients mostly developed only after levodopa initiation. Overall, rotigotine transdermal patch has demonstrated favorable clinical efficacy and tolerability in the treatment of PD and RLS.

Continuous rotigotine administration reduces dyskinesia resulting from pulsatile treatment with rotigotine or L-DOPA in MPTP-treated common marmosets.

We previously showed that continuous infusion of rotigotine resulted in less dyskinesia than repeated pulsatile rotigotine administration or repeated oral administration of L-DOPA in MPTP-treated marmosets. Now we investigate whether continuous rotigotine delivery modifies established dyskinesia induced by prior exposure to repeated pulsatile administration of L-DOPA or rotigotine in MPTP-treated common marmosets. Repeated oral administration of L-DOPA or subcutaneous bolus administration of rotigotine over 28 days improved motor deficits but resulted in the onset of dyskinesia of moderate intensity. When these animals were switched to a 28-day continuous infusion of rotigotine, the reversal of motor disability was maintained. In those animals initially treated with L-DOPA, there was a small reduction in dyskinesia intensity but a significant reduction in the duration of dyskinesia. However, in animals initially treated with repeated bolus administration of rotigotine, dyskinesia intensity was significantly reduced. Initial treatment with a continuous infusion of rotigotine for 28 days reversed motor disability and resulted in a low incidence of dyskinesia. On switching to repeated oral administration of L-DOPA, the improvement in motor disability was maintained but the propensity of L-DOPA to provoke dyskinesia was not affected. In addition, while the continuous delivery of rotigotine prevented the expression of dyskinesia, the previously demonstrated ability of dopamine agonists to prime for dyskinesia could not be avoided. These data suggest that dyskinesia induced by pulsatile drug treatment may be improved by switching to continuous rotigotine delivery. In addition, while continuous delivery of rotigotine may prime for dyskinesia, it does not lead to its expression.

Continuous administration of rotigotine to MPTP-treated common marmosets enhances anti-parkinsonian activity and reduces dyskinesia induction.

Rotigotine is a novel, non-ergoline dopamine D(3)/D(2)/D(1)-receptor agonist for the treatment of Parkinson's disease that can be continuously delivered by the transdermal route to provide stable plasma levels. Continuous drug delivery should reduce the risk of dyskinesia induction in comparison to pulsatile dopaminergic treatment. Thus the aim of the study was to compare the reversal of motor disability and induction of dyskinesia produced by continuous compared to pulsatile rotigotine administration in MPTP-treated common marmosets. The study also investigated whether pulsatile or continuous rotigotine administration in combination with l-DOPA prevented l-DOPA-induced dyskinesia. Animals were treated for 28 days with vehicle or pulsatile (twice daily) or continuous delivery of rotigotine (via an osmotic minipump). Subsequently, l-DOPA was then co-administered for a further 28 days. Animals were assessed for locomotor activity, motor disability and dyskinesia induction. The study showed that both continuous and pulsatile administration of rotigotine improved motor deficits and normalized motor function in MPTP-treated monkeys. However, continuous rotigotine delivery reduced dyskinesia expression compared to pulsatile treatment. Both pulsatile and continuous rotigotine administration produced less dyskinesia than administration of l-DOPA alone. The addition of l-DOPA to either pulsatile or continuous rotigotine treatment resulted in the induction of marked dyskinesia similar to that produced by treatment with l-DOPA alone. These data further support the hypothesis that continuous delivery of a dopaminergic agent reduces the risk of dyskinesia induction. However, continuous rotigotine administration did not prevent l-DOPA from inducing dyskinesia suggesting that l-DOPA may induce dyskinesia by mechanisms different from dopamine agonist drugs.

Continuous delivery of rotigotine decreases extracellular dopamine suggesting continuous receptor stimulation.

Rotigotine, a non-ergolinic dopamine receptor agonist for treatment of Parkinson's disease was continuously administered over 48 h (0.5 mg/kg s.c., slow release formulation) to conscious rats striatally implanted with a microdialysis probe. Subsequently, the levels of rotigotine increased to a maximum of 3.42 + 2.1 nmol/l and remained at a level of 2.81 +/- 0.82 nmol/l for 48 h. Concomitantly, the dopamine levels consistently decreased to 20% of the control level. This suggests that the sustained administration of rotigotine provides stable extracellular drug levels in the striatum resulting in continuous stimulation of dopamine receptors.

The lateral spread of epileptiform discharges in rat neocortical slices: effect of focal phencyclidine application.

In vitro and in vivo brain slice techniques were used to examine phencyclidine (PCP) effects on the lateral propagation of epileptiform field potentials (EFP) across adjacent areas of rat frontal neocortex. Epileptiform activity was induced by perfusing slices with Mg 2+-free artificial cerebrospinal fluid. Simultaneous field potential recordings of EFP were obtained from four microelectrodes placed 2-3 mm apart across coronal slices in the third layer. PCP, applied focally between recording sites, blocked rapid propagation across treated areas and resulted in the emergence of spatially separate, independent pacemakers. The characteristics of paroxysmal depolarization shifts did not change significantly by the blockade of lateral propagation of EFP. The same asynchronized pattern of EFP conduction was observed after local application of the N-methyl-D-aspartate (NMDA)-receptor antagonist DL-2-amino-5-phosphono-valeric acid. Local administration of haloperidol as well as NMDA before PCP application reversibly prevented appearance of multiple pacemakers. Focal application of dopamine produced an abnormal pattern of lateral conduction of EFP in 50 % of tested slices. Pacemaker failure as an indicator of functional impairment of cortical integration is the proposed mechanism for developing of schizophrenia-like psychosis associated with epilepsy. Abbreviations. APV: DL-2-amino-5-phosphono-valeric acid EEG:electroencephalogram EFP:epileptiform field potentials NMDA:N-methyl-D-aspartate PCP:phencyclidine SLPE:Schizophrenialike psychosis associated with epilepsy

[Prospective comparison of hydrosonography, endosonography and specimen sonography for TN staging of gastric carcinoma]. / Prospektiver Vergleich von Hydrosonographie, Endosonographie und Präparatesonographie beim TN-Staging von Magenkarzinomen.

PURPOSE: To compare hydrosonography (HUS), endosonography (EUS) and experimental sonography (PUS) with respect to TN-staging accuracy of gastric carcinoma. MATERIAL AND METHODS: Thirty-six patients with gastric carcinoma underwent EUS (7.5/12 MHz transducer, Olympus GF-UM 20) and HUS (3.75 MHz transducer, Toshiba, Sonolayer SSA-270A) for TN-staging according to the UICC-classification. The resected specimens were reexamined (3.75/7.5 MHz transducer) and again TN-staging was performed. Findings were correlated with histopathological results. RESULTS: T- and N-staging accuracies were as follows: EUS 54 % (19/35) and 79 % (27/34); HUS 41 % (15/37) and 61 % (22/36); and PUS 51 % (19/37) and 72 %(26/36). Sensitivities and specificities for the detection of lymph node metastases were as follows: EUS 87 % and 54 %; HUS 57 % and 69 %; and PUS 83 % and 54 %. CONCLUSIONS: The accuracy of sonographic TN- staging is limited in patients with gastric carcinoma. Nevertheless, EUS may contribute to the preoperative management of patients with gastric carcinoma if indications are well defined. HUS is not suited for TN-staging of gastric carcinoma.

Spreading depression in human neocortical slices.

Cortical spreading depression (CSD) occurrence has been suggested to be associated with seizures, migraine aura, head injury and brain ischemia-infarction. Only few studies identified CSD in human neocortical slices and no comprehensive study so far evaluated this phenomenon in human. Using the neocortical tissue excised for treatment of intractable epilepsy, we aimed to investigate CSD in human. CSD was induced by KCl injection and by modulating T-type Ca(2+) currents in incubated human neocortical tissues in an interphase mode. The DC-fluctuations were recorded by inserting microelectrodes into different cortical layers. Local injection of KCl triggered single CSD that propagated at 3.1+/-0.1 mm/min. Repetitive CSD also occurred spontaneously during long lasting application (5 h) of the T-type Ca(2+) channel blockers amiloride (50 microM) or NiCl(2) (10 microM) which was concomitant with a reversible extracellular potassium increase up to 50 mM. CSD could be blocked by the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid in all cases. The results demonstrate that modulation of the Ca(2+) dynamics conditioned human neocortical slices and increased their susceptibility to generate CSD. Furthermore, these data indicate that glutamatergic pathway plays a role in CSD phenomenon in human.

Therapeutic drug monitoring of tacrolimus in pediatric liver transplant patients.

The clinical utility of tacrolimus monitoring in adults has been well documented. The present study compared tacrolimus monitoring in a pediatric population of 34 liver transplant patients in four US centers with an adult population of 111 patients in six US centers. Subjects (adult and pediatric) were evaluated, at defined intervals over 12 weeks post-transplantation (Tx), for tacrolimus trough concentrations and 12 additional laboratory chemistries. Pediatric patient and graft survival for the 12 weeks were 91% and 88%, respectively, as compared to 97% and 93%, respectively, for the adult population. The mean oral dosage of tacrolimus for pediatric patients was 0.13 +/- 0.1 mg/kg/day at week 1, increased to 0.30 +/- 0.3 mg/kg/day by week 3 and remained constant for the remainder of the study. These dosages were two- to three-fold higher than the dosage used in the adult population. In contrast, the mean whole-blood trough concentration, as determined by PRO-Tractrade mark II enzyme-linked immunosorbent assay (ELISA), decreased from 11.3 +/- 5.1 ng/mL at week 1 to 6.3 +/- 3.7 ng/mL by week 12 and was not significantly different from the trough concentration in adults. The incidence and distribution of the clinical end-points for the pediatric subjects (rejection, nephrotoxicity, death, re-Tx) were different from those observed in adults. The total percentage of pediatric subjects reaching any end-point was 74%, as compared to 54% in the adult population. These data indicate several differences between the adult and pediatric populations in their response to tacrolimus.

Clinical utility of monitoring tacrolimus blood concentrations in liver transplant patients.

The relationship between the dose of tacrolimus, trough tacrolimus blood concentration, and selected clinical endpoints (acute rejection, nephrotoxicity, and other toxicities) were examined in a prospective, multicenter clinical trial to validate the use of an enzyme-linked immunosorbent assay (ELISA) for monitoring whole-blood concentrations of tacrolimus in liver transplant patients. A total of 111 subjects from six transplant centers were evaluated over 12 weeks posttransplantation. In addition to trough tacrolimus blood concentrations, hematocrit, ALT, AST, GGTP, alkaline phosphatase, total bilirubin, serum creatinine, BUN, serum potassium, serum magnesium, blood glucose, and serum albumin were also measured. The relationship between trough tacrolimus blood concentrations and clinical endpoints was analyzed using both a logistic regression model and a Cox proportional hazard model. By logistic regression analysis, a statistically significant (p = 0.0465) relationship between increasing trough tacrolimus blood concentrations and decreasing risk of acute rejection was demonstrated over a 7-day time window. Nephrotoxicity and other toxicities also demonstrated statistically significant relationships with trough tacrolimus blood concentrations. The results of the Cox analysis were consistent with the logistic regression analysis. Using receiver operator characteristic curves, trough tacrolimus concentrations as measured by the ELISA method were able to differentiate the occurrence of nephrotoxicity and toxicity from nonevents. To minimize nephrotoxicity of tacrolimus, it is necessary to maintain trough blood concentrations below 15 ng/ml. This study demonstrates that the ELISA method used to measure tacrolimus blood concentrations in this study provides information of predictive value for managing the risk of nephrotoxicity, other toxicity, and rejection in liver transplant patients.

Cerebral taurine release mechanisms in vivo: pharmacological investigations in rats using microdialysis for proof of principle.

Cerebral taurine acts as neurotransmitter, as neuromodulator, or as osmoregulator. To investigate its release mechanisms in vivo, we combined the microdialysis technique with a variety of experimental paradigms. Taurine release was stimulated by either NMDA, NO or a hypotonic solution locally with or without the addition of the NMDA antagonists APV or Ketamine, or the NO synthase inhibitor L-NAME. Alternatively, the neuroprotective drug lubeluzole was applied i.v. NMDA, NO or the hypotonic solution stimulated the release of taurine. NMDA-mediated taurine release was inhibited by either APV, Ketamine or the NO synthase inhibitor L-NAME. Lubeluzole had no effect. Under the hypotonic conditions only lubeluzole was effective. These data confirm in vivo that the NMDA-induced taurine release is mediated via the NO cascade. By contrast, the release after a hypotonic stimulus is not related to the NO cascade. Instead, Na(+)- and/or Ca(2+)-mediated events might have been attenuated by lubeluzole.

NiCl2 and amiloride induce spreading depression in guinea pig hippocampal slices.

Spreading depressions (SD) occur in association with ischaemia, epilepsy and migraine. Intracellular calcium oscillations have been suggested to be involved in the generation and propagation of SD. The present study was performed to study the mechanism of conditioning guinea pig hippocampal slices by the T-type calcium channel blockers NiCl2 and amiloride. SD-like fluctuations of DC potential were recorded by inserting microelectrodes into the CA1 and CA3 regions. The SD occurrence was significantly greater with 10 micromol/l NiCl2 as well as with 25 and 50 micromol/l amiloride than with other concentrations of these substances. The concentration response curve was inversely U-shaped with the maximum repetition rates of SDs being achieved at 10 micromol/l NiCl2 as well as at 25 and 50 micromol/l amiloride. SD occurrence could be completely blocked by the NMDA antagonist APV (10 micromol/l) in all cases. These data demonstrate that modulation of the Ca2+ dynamics conditioned guinea pig hippocampal slices and increased their susceptibility to generate SD.

Observations on the relationship between the extracellular changes of taurine and glutamate during cortical spreading depression, during ischemia, and within the area surrounding a thrombotic infarct.

Taurine and glutamate were monitored by microdialysis technique during various cerebral insults: a. Application of K+ triggered a cortical spreading depression (CSD). Taurine and glutamate increased concomitantly but recovery of glutamate was faster than that of taurine. b. Application of NMDA induced also CSD but only taurine increased. c. Induction of an infarct triggered repetitive CSDs. Taurine increased rapidly whereas glutamate rose slowly starting with some delay. d. After induction of ischemia, taurine and glutamate increased after onset of depolarisation. The increase of glutamate occurred late after a small, transient increase in parallel with the depolarisation. These data suggest a close functional relationship between the changes of both amino acids. Therefore, they should be monitored together especially in clinical settings: during excitation, only taurine will increase; during overexcitation, taurine will also increase but to a higher maximum followed by a moderate rise of glutamate; after energy failure, taurine will accumulate to its highest level followed by a continuous rise of glutamate.

Neurochemical changes and laser Doppler flowmetry in the endothelin-1 rat model for focal cerebral ischemia.

Generalized neurotransmitter overflow into the extracellular space, after cerebral ischemia, has been suggested to contribute to subsequent neuronal death. This study aims to investigate the striatal release of the neurotransmitters dopamine (DA), glutamate (Glu) and gamma-aminobutyric acid (GABA) by means of microdialysis, in a rat model for focal transient cerebral ischemia. Ischemia was induced by the application of 120 pmol endothelin-1 (Et-1), adjacent to the middle cerebral artery (MCA) in freely moving rats. Ischemia produced a large increase in extracellular striatal DA concentrations (2400%), Glu (5500%) and GABA (800%) concentrations. Laser Doppler flowmetry in anaesthetized rats, indicated that the blood flow within the striatum decreased by 75+/-11%. The period of sustained drop of blood flow, was dose-dependently related to the concentration Et-1 injected. Histological analysis of brain slices, taken from anaesthetized and conscious animals, indicated a 500 pmol dose of Et-1 was required to produce a similar infarct in anaesthetized rats to a 120 pmol dose of Et-1 in freely moving rats. The immediate drop in striatal blood flow, and the prompt increase of extracellular DA, after the micro-application of Et-1, were quite striking. This suggests that the DA release, rather than the Glu overflow may be the primary event initiating the cascade of processes ultimately leading to cell death and neurological deficits.

Experimental and theoretical 31P and 77Se nuclear magnetic shielding tensors for bis(dineopentoxyphosphorothioyl) diselenide.

An intergrown crystal of two phases of bis(dineopentoxyphosphorothioyl) diselenide 1 was investigated by goniometer 31P NMR. From the angular dependence of the chemical shift, the tensors of a triclinic and a monoclinic phase were determined. The principal values sigma11, sigma22, and sigma33, of the absolute nuclear magnetic shielding tensors for the triclinic phase are 134.1, 227.2, and 375.5 ppm and for the monoclinic phase are 132.4, 227.8, and 374.2 ppm, respectively. In both cases, the principal axis 3 of the 31P tensor is directed nearly along the P=S bond and the principal axis 2 is nearly perpendicular to the S=P-Se plane. Calculations of the 31P and 77Se nuclear magnetic shielding tensors were performed for molecules of both phases of 1 and for model compounds by the sum-over-states density functional perturbation theory IGLO method. The rms distances between calculated and experimental 31P NMR icosahedral tensor values sigma(j) (j = 1, ..., 6) amount to 17-21 ppm. The calculated and experimental orientations of the 31P principal axes show a maximum difference of 5 degrees and rms distances of 3.2 and 3.3 degrees. For the principal value sigma33 of the selenium shielding tensor the agreement between calculated and experimental values is satisfactory, but the calculated values sigma11 and sigma22 are distinctly too small. Calculations for a model compound in which the methyl groups of the neopentoxy residue are substituted by protons lead practically to the same results.

Extracellular taurine as a parameter to monitor cerebral insults 'on-line': time courses and mechanisms as studied in vivo.

UNLABELLED: Taurine increases in the zone surrounding a thrombotic infarct which could be prevented by a neuroprotective drug. Therefore, we aimed at studying the possible release mechanisms since the monitoring of taurine might give valuable information on the progress of cerebral insults and the effect of drugs. A microdialysis membrane was implanted into the cortex of anaesthetised rats: As toxic triggers possibly released by the dying cells in the peri-infarct zone, either a glutamatergic agonist (NMDA) or high potassium were applied via the microdialysis probe. Alternatively, a diluted perfusate was applied to induce cell swelling directly. NMDA antagonists or the NO synthase inhibitor L-NAME were applied locally too. NMDA, NO, high potassium or the hypotonic solution stimulated the release of taurine. The effect of high potassium could be prevented by Ketamine, but not by APV. The effect of NMDA could be inhibited by APV or Ketamine or the NO synthase inhibitor L-NAME. The release of taurine induced by the hypotonic solution could not be be reduced by any of the inhibitors. These data suggest that the release of taurine induced by glutamatergic activity is mediated via the NO cascade. The potassium mediated release seems to be related only in part to glutamatergic activity. Thus, other mechanisms seem to be predominate in potassium mediated swelling. Hypoosmotically induced taurine release is not mediated via the NO cascade and also seems to differ from the aforementioned release mechanisms. IN CONCLUSION: Monitoring of extracellular taurine allows to follow pathological events and to differentiate drug effects.

Analysis of whole blood tacrolimus concentrations in liver transplant patients exhibiting impaired liver function.

In transplant patients with impaired liver function, HPLC methodologies have been suggested for monitoring whole blood tacrolimus concentrations because of the reported inaccuracy of immunoassay for whole blood tacrolimus concentrations. One hundred fifty whole blood samples from 50 subjects enrolled in a multicenter liver transplant trial were chosen for HPLC/MS/MS analysis without consideration of the clinical status of the patient at the time of sampling. These samples were chosen to represent the sampling intervals during the 12-week posttransplantation period. Retrospectively, the authors identified a subset of 39 samples from 27 subjects exhibiting impaired liver function as demonstrated by bilirubin concentrations > 3.0 mg/dL (mean +/-SD = 7.5 +/- 5.6 mg/dL). The authors compared the agreement of concentrations obtained from the PRO-Trac II ELISA and HPLC/MS/MS by least squares linear regression analysis and Bland/Altman analysis, in this subset against the agreement of concentrations for 76 samples with normal bilirubin. In the samples obtained from patients with impaired liver function the resulting regression equation was: ELISA = 1.19(HPLC) + 0.7; r = 0.9. The mean difference (HPLC/MS/MS - ELISA) was -2.5 ng/mL +/- 2.9 ng/mL (mean +/- SD). While 71% of samples agreed within 3 ng/mL, 3% (n = 1) exhibited a difference >10 ng/ml. The corresponding evaluation of the samples with normal bilirubin concentrations resulted in the regression equation ELISA = 0.96(HPLC) + 0.9; r = 0.9, and a mean difference of -0.6 ng/mL +/- 2.3 ng/mL. The authors conclude that while a small subset of patients with cholestasis may require closer evaluation with a more specific methodology, the majority of the patients may be satisfactorily monitored with the PRO-Trac II ELISA.

[Redox sensitive behavior of selenite in the presence of reactive oxygen species. Are there nonenzymatic direct reaction pathways]. / Zum redoxsensitiven Verhalten von Selenit in Gegenwart reaktiver Sauerstoffspezies. Gibt es nichtenzymatische direkte Reaktionswege?

From extensive research over the last decade it has been known that selenium is essential as necessary component of selenoaminoacids and of specific enzymes. Among others, the redoxpair GSH/GSSG is closely connected with antioxidative processes. Moreover it inhibits and/or activates molecular key reactions with the involvement of various small reactive O- and N-species. We investigated the direct interaction of selenite with components of the respiratory burst of human blood cells, considering the redoxamphoterie of alkali-selenite. Selenite tend to redox-disproportation depending on the pH-value. Whether selenite leads to oxidation or reductation is dependent not only on the pH-value, but also on the redox-potential of the reaction partners. In in-vivo adapted in-vitro conditions (ph = 7.4; mumolar concentrations of reaction partners) we observed the following results: 1. SeO3(2-) is not oxidized by H2O2/NO or triplet-oxygen, when the oxidatives are applied alone; 2. SeO3(2-) is quantitatively oxidized from SeO4(2-) by the combination H2O2/NO2- or O2-/NO; 3. SeO3(2-) is semiquantitatively oxidized by singlett oxygen to SeO4(2-). The composition of reaction products was measured by 77Se-NMR-spectroscopy. The reactive intermediate product for the 2. reaction should be peroxynitrite (HOONO). One cannot rule out the possibility that HOONO reacts on a large scale with H2O2 to singlett oxygen. Subsequently singlett oxygen oxidizes selenite. The pathophysiological impact of singlett oxygen in processes like arteriosclerosis is now being investigated. It has been supposed, that singlett oxygen is participating in processes of lipidperoxidation invivo. Further investigations have to show, to what extent selenite is effective as direct 1O2-scavanger.