Isoprenoidquantifizierung in Hirngewebe - cerebrale Regulation von FPP und GGPP bei Morbus Alzheimer und im Alter

Over the last years there has been an increasing interest in the involvement of the MVA-pathway and of members of the small GTPases, in the development and progression of AD. Earlier investigations mainly focused on the role of cholesterol in disease pathology. This research was supported by retrospective cohort studies, initially showing beneficial effects of the long-term intake of cholesterol lowering statins, on the incidence of the development of sporadic AD. However, in more recent literature increasing attention has been paid to the isoprenoids, FPP and GGPP, due to their crucial role in the post-translational modifications of members of the superfamily of small GTPases. In AD, these proteins were amongst others shown to be involved in mechanisms affecting APP processing, ROS generation and synaptic plasticity. A major factor impeding the clarification of the role of the MVA-pathway intermediates in these mechanisms was the lack of a sensitive and accurate method to determine FPP and GGPP levels in brain tissue. Hence, a state of the art HPLC-FLD method for the quantification of the isoprenoids FPP and GGPP in brain tissue was successfully developed. After the introduction of a double clean-up step from complex brain matrix samples and the synthesis of an appropriate IS (DNP), the method was fully validated according to the latest FDA guideline for bioanalytical method validation. Furthermore, this method was transferred to a faster and more sensitive, state of the art UHPLC-MS/MS application. Additionally, the method was shown to be applicable for mouse brain tissue and data was generated from an in vivo mouse simvastatin study and for different mouse models. According to the aims of the thesis, the current work describes for the first time absolute isoprenoid concentrations in human frontal cortex white and grey matter. Furthermore, this is the first report of isoprenoid levels in the frontal cortex of human AD brains. Further results were shown from mouse brains originating from different mouse models, including the Thy-1 APP mouse model mimicking AD pathology in terms of Aβ formation or C57Bl/6 mice at different ages. AD prevalence can be clearly correlated with increasing age. Therefore, three different generations of mice were investigated. The study demonstrated constant isoprenoid and cholesterol levels in the first half of their life followed by a significant increase of FPP and GGPP in the second half (between 12 and 24 month of age). Cholesterol levels were also elevated in the aged group, but again the effect was less pronounced than shown for the isoprenoids. These results lead to the tentative conclusion that cerebral isoprenoid levels are elevated during aging and that this accumulation is amplified during AD leading to accelerated neuronal dysfunction. In a different mouse study, using the C57Bl/6 mice, in vivo drug intervention with the HMG-CoA reductase inhibitor simvastatin revealed strong inhibition of the rate limiting step of the mevalonate/isoprenoid/cholesterol pathway and resulted in the first report of significantly reduced FPP and GGPP levels in brain tissue of statin treated mice. These results open for the first time the possibility to monitor drug effects on cerebral isoprenoid levels and correlate these data with a modulation of APP processing, which was shown by our group in previous studies. Interestingly, apart from the isoprenoid reduction following statin treatment the reduction of brain cholesterol was also significant but to a lesser extent. These findings support the notion that isoprenoid levels are more susceptible to statin treatment than cholesterol levels. Furthermore, this suggests a strong cellular dependence on FPP and GGPP, as the pool seems to be easily depleted, which finally could lead to cell death. The first investigations of farnesylated Ras and geranylgeranylated Rac protein levels by means of immuno-blotting, substantiated the notion of a decreased abundance of prenylated small GTPases under statin influence as a consequence of reduced isoprenoid levels. These findings demonstrate for the first time a correlation of FPP and GGPP levels with the abundance of small GTPases. These findings together with the results from the AD study prove that isoprenoid levels are not strictly subject to the same regulation as cholesterol levels. To further understand the physiological regulation in the cell, in vitro experiments with different inhibitors of the mevalonate/isoprenoid/cholesterol pathway were conducted. These results confirmed the isoprenoid and cholesterol reducing effects of statin treatment as observed in the aforementioned in vivo mouse study. Interestingly, cholesterol synthesis inhibition targeted after FPP as the branch point, led to significantly elevated FPP levels. FTase inhibition led to significantly reduced FPP levels, whereas inhibition of the GGTase I did not show a significant change of either isoprenoid levels.Im Zusammenhang mit der Entstehung und dem Fortschreiten der Alzheimer Demenz (AD) spielt speziell der Mevalonat-Biosyntheseweg eine bedeutsame Rolle. Frühere Arbeiten beschäftigten sich hauptsächlich, gestützt durch die Ergebnisse retrospektiver Studien mit Statinen, mit der Rolle des Cholesterins. Aktuellere Arbeiten richten den Fokus der Forschung stärker auf die ebenfalls aus dem Mevalonat-Biosyntheseweges entstammenden Isoprenoide, Farnesyl- (FPP) und Gernaylgeranylpyrophosphat (GGPP). Beide Isoprenoide sind maßgeblich an der post-translationalen Modifikation von Proteinen aus der Familie der kleinen Rho-GTPasen beteiligt. Bezüglich der AD konnte gezeigt werden, dass bestimmte Mitglieder dieser Familie in Mechanismen involviert sind die an der Entstehung von reaktiven Sauerstoffspezies und neurotoxischem Amyloid beta beteiligt sind. Weiterhin spielen sie bei der synaptischen Plastizität eine zentrale Rolle. Bisherige Forschungen zur Aufklärung der genauen biochemischen Funktion von FPP und GGPP sowie deren Regulation, speziell in Hirngewebe, wurden durch das Fehlen einer sensitiven und validen Analytik eingeschränkt. Die vorgelegte Arbeit beschreibt die Entwicklung, Validierung und erfolgreiche Anwendung einer HPLC-Fluoreszenz Methode zur Quantifizierung der beiden Isoprenoide FPP und GGPP in Hirngewebe. Der Erarbeitung eines komplexen Protokolls zur Methodenaufarbeitung und der Synthese eines geeigneten internen Standards folgte eine komplette Validierung nach den aktuellen FDA Richtlinien für bioanalytische Methodenvalidierung. Des Weiteren wurde diese Methode auf eine schnellere, sensitivere und selektivere, hoch-moderne UHPLC-MS/MS Methode übertragen und neben der Validierung der Methode für humane Hirnproben wurde auch die Übertragbarkeit auf Mäusehirne gezeigt. Entsprechend der Zielsetzung dieser Arbeit wurden erstmalig FPP und GGPP Konzentrationen in der weißen und grauen Substanz des menschlichen frontalen Kortex quantifiziert. Weiter konnten unter Verwendung der neu etablierten Methode zum ersten Mal erhöhte FPP und GGPP Spiegel im frontalen Kortex von AD Patienten verglichen mit gleichaltrigen Kontroll-Hirn Proben nachgewiesen werden. Für die Cholesterin Spiegel der identischen Proben konnten gezeigt werden, dass diese unverändert waren, was frühere Arbeiten bestätigt. Die vorgelegte Arbeit liefert weiterhin neue Ergebnisse zu der Korrelation von cerebralen Isoprenoid- und Cholesterin-Spiegeln in einem in vivo Thy-1 APP Maus-Model, welches typischer Weise herangezogen wird um den, der AD zugrunde liegenden Amyloid beta Metabolismus zu untersuchen. Weiter wurden Studien an einem Alterungsmodell mit C57Bl/6 Mäusen durchgeführt, die eine zunächst (zwischen Mäusen im Alter zwischen 3 und 12 Monaten) unveränderte FPP und GGPP Homöostase zeigten, während in der dritten und ältesten Gruppe (24 Monate) ein signifikanter Anstieg zu verzeichnen war. Diese Ergebnisse wurden auch durch die gemessenen Cholesterin Spiegel reflektiert, die ebenfalls nur im Alter erhöht waren. Da das Auftreten der AD deutlich mit dem Alter korreliert führten diese Ergebnisse, zusammen mit den Daten von den humanen AD Hirnproben zu der Hypothese, dass Isoprenoid Spiegel mit dem Alter im Gehirn ansteigen und dieser Effekt bei der AD möglicherweise potenziert ist. In weiteren in vivo Versuchen mit C57Bl/6 Mäusen konnte erstmalig der pharmakologische Effekt von Simvastatin auf die Gehalte von FPP und GGPP im Gehirn gezeigt werden. Die orale Verabreichung des HMG-CoA Reduktase Inhibitors für 21 Tage führte zu stark reduzierten FPP und GGPP Konzentrationen im Gehirn der behandelten Tiere. Cholesterin wurde durch die Behandlung ebenfalls reduziert, wobei dieser Effekt vergleichsweise schwach ausgeprägt war. Dies lässt darauf schließen, dass Isoprenoid-Spiegel sensibler auf eine Hemmung der HMG-CoA Reduktase reagieren als Cholesterin-Spiegel. Mevalonat-Biosyntheseweges als Mevalonat/Isoprenoid/Cholesterin-Biosyntheseweg. Um weitere Einblicke in die physiologische Regulation dieses speziellen Biosyntheseweges zu erlangen, wurden einerseits in mehreren Kooperationen eine ganze Reihe von weiteren Metaboliten des Mevalonat-Biosyntheseweges an dem zuvor beschriebenen in vivo Alterungsmodels untersucht (Daten noch in Bearbeitung). In der Gesamtheit wurden, wie oben beschrieben bei FPP und GGPP die stärksten Veränderungen im hohen Alter festgestellt, was die bedeutende Rolle der beiden Isoprenoide in diesem Biosyntheseweg unterstreicht. Andererseits wurden im Zellmodel (SY5Y Zellen) unterschiedliche Inhibitoren des Mevalonat/Isoprenoid/Cholesterin-Biosyntheseweges untersucht. Hierbei konnte der Statin-Effekt aus dem in vivo Versuch ebenfalls in vitro gezeigt werden. Inhibition der Squalensynthase, die den Cholesterin-Biosyntheseweg nach der FPP Synthesestufe hemmt, führte zu signifikant erhöhten FPP Spiegel, was auf eine Akkumulation des Isoprenoids hindeutet

Dried blood spot UHPLC-MS/MS analysis of oseltamivir and oseltamivircarboxylate—a validated assay for the clinic

The neuraminidase inhibitor oseltamivir (Tamiflu®) is currently the first-line therapy for patients with influenza virus infection. Common analysis of the prodrug and its active metabolite oseltamivircarboxylate is determined via extraction from plasma. Compared with these assays, dried blood spot (DBS) analysis provides several advantages, including a minimum sample volume required for the measurement of drugs in whole blood. Samples can easily be obtained via a simple, non-invasive finger or heel prick. Mainly, these characteristics make DBS an ideal tool for pediatrics and to measure multiple time points such as those needed in therapeutic drug monitoring or pharmacokinetic studies. Additionally, DBS sample preparation, stability, and storage are usually most convenient. In the present work, we developed and fully validated a DBS assay for the simultaneous determination of oseltamivir and oseltamivircarboxylate concentrations in human whole blood. We demonstrate the simplicity of DBS sample preparation, and a fast, accurate and reproducible analysis using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer. A thorough validation on the basis of the most recent FDA guidelines for bioanalytical method validation showed that the method is selective, precise, and accurate (≤15% RSD), and sensitive over the relevant clinical range of 5–1,500 ng/mL for oseltamivir and 20–1,500 ng/mL for the oseltamivircarboxylate metabolite. As a proof of concept, oseltamivir and oseltamivircarboxylate levels were determined in DBS obtained from healthy volunteers who received a single oral dose of Tamiflu®

Analytical Investigations of Toxic p-Phenylenediamine (PPD) Levels in Clinical Urine Samples with Special Focus on MALDI-MS/MS

Para-phenylenediamine (PPD) is a common chromophoric ingredient in oxidative hair-dyes. In some African countries like Sudan, Egypt and Morocco but also in India this chemical is used alone or in combination with colouring extracts like Henna for dyeing of the hair or the skin. Excessive dermal exposure to PPD mainly leads to the N-mono- and N,N′-diacetylated products (MAPPD, DAPPD) by N-acetyltransferase 1 and 2 (NAT1 and 2) catalyzed reactions. Metabolites and PPD are mainly excreted via renal clearance. Despite a low risk of intoxication when used in due form, there are numerous cases of acute intoxication in those countries every year. At the ENT Hospital - Khartoum (Sudan) alone more than 300 cases are reported every year (∼10% fatal), mostly caused by either an accidental or intended (suicidal) high systemic exposure to pure PPD. Intoxication leads to a severe clinical syndrome including laryngeal edema, rhabdomyolysis and subsequent renal failure, neurotoxicity and acute toxic hepatitis. To date, there is no defined clinical treatment or antidote available and treatment is largely supportive. Herein, we show the development of a quick on-site identification assay to facilitate differential diagnosis in the clinic and, more importantly, the implementation of an advanced analytical platform for future in-depth investigations of PPD intoxication and metabolism is described. The current work shows a sensitive (∼25 µM) wet chemistry assay, a validated MALDI-MS/MS and HPLC-UV assay for the determination of PPD and its metabolites in human urine. We show the feasibility of the methods for measuring PPD over a range of 50–1000 µM. The validation criteria included linearity, lower limit of quantification (LLOQ), accuracy and precision, recovery and stability. Finally, PPD concentrations were determined in clinical urine samples of cases of acute intoxication and the applied technique was expanded to identify MAPPD and DAPPD in the identical samples

Incurred sample reanalysis comparison of dried blood spots and plasma samples on the measurement of lopinavir in clinical samples

Background: Undoubtedly, incurred sample reanalysis (ISR) will become an integral part of regulated bioanalysis of dried blood spot (DBS) samples. In this article, we report results from an ISR study on DBS specimen and their corresponding plasma samples. Incurred samples were reanalyzed on their concentration of the antiretroviral drug lopinavir (LPV). Results: Bland-Altman comparison plots showed a high degree of agreement between the measurements; 94.7% of observed LPV concentrations were within bias +/- 2 SD. Moreover, 73.7% of obtained LPV concentrations from DBS ISR were in good compliance with general acceptance criteria (4-6-20 rule) on ISR testing, while plasma ISR failed on these acceptance criteria due to the low compliance of 10.5%. Conclusion: It was demonstrated that plasma ISR testing failed on acceptance criteria while corresponding incurred DBS specimens passed. Furthermore, the current article demonstrates that the stability of the antiretroviral drug LPV was significantly different in both biological matrices

Dried matrix on paper disks: the next generation DBS microsampling technique for managing the hematocrit effect in DBS analysis

Background: The hematocrit effect is a hurdle for successful introduction of the dried blood spot (DBS) in a regulated environment. Recently, attempts were taken to overcome the hematocrit effect by whole-cut DBS analysis. This paper presents the next-generation whole-cut DBS; dried matrix on paper disks (DMPD). Results: DMPD eliminated the hematocrit effect and demonstrated better accuracy and precision than regular DBS with partial punching. Observed accuracy and precision were 6.0 and 2.3% for DMPD, respectively, and -10.4 and 17.1%, for DBS, respectively. Conclusion: The DMPD technique performed better than regular DBS by eliminating the hematocrit effect related blood volume bias. Although this effect was not observed with DMPD, a systematic error of 6.0% was detected and further technical development of DMPD could improve the performance

Calibration curve of PPD (A) on the MALDI-MS/MS system (50–1000 µmol/L) and (B) on the HPLC-UV system (150–1000 µmol/L).

<p>Graphs shows the mean values of (<b>A</b>) 5 and (<b>B</b>) 3 independent measurements and the corresponding <i>r²</i> value. Mean +/− SEM.</p

Validation results of PPD in human urine.

<p>Inter- and intraday accuracy and precision measurements (<i>n = 3</i> for each value). The mean relative error of each concentration is expressed as % bias and the reproducibility is depicted as the coefficient of variation (% CV). Intraday accuracy and precision were also determined on the HPLC-UV system.</p

Validation results of PPD in human urine.

<p>MALDI-MS/MS assay: linearity, lower limit of quantification (LLOQ), recovery (at 130, 400 and 800 µmol/L) and stability after addition of formic acid.</p