Characterization of ß-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry.

Plasmid-encoded ß-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the ß-lactam ring structure of certain ß-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of ß-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine ß-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the ß-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible ß-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of ß-lactamase activity in various bacterial strains by mass spectrometry.

Isoprenoids, small GTPases and Alzheimer's disease.

The mevalonate pathway is a crucial metabolic pathway for most eukaryotic cells. Cholesterol is a highly recognized product of this pathway but growing interest is being given to the synthesis and functions of isoprenoids. Isoprenoids are a complex class of biologically active lipids including for example, dolichol, ubiquinone, farnesylpyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP). Early work had shown that the long-chain isoprenoid dolichol is decreased but that dolichyl phosphate and ubiquinone are elevated in brains of Alzheimer's disease (AD) patients. Until recently, levels of their biological active precursors FPP and GGPP were unknown. These short-chain isoprenoids are critical in the post-translational modification of certain proteins which function as molecular switches in numerous signaling pathways. The major protein families belong to the superfamily of small GTPases, consisting of roughly 150 members. Recent experimental evidence indicated that members of the small GTPases are involved in AD pathogenesis and stimulated interest in the role of FPP and GGPP in protein prenylation and cell function. A straightforward prediction derived from those studies was that FPP and GGPP levels would be elevated in AD brains as compared with normal neurological controls. For the first time, recent evidence shows significantly elevated levels of FPP and GGPP in human AD brain tissue. Cholesterol levels did not differ between AD and control samples. One obvious conclusion is that homeostasis of FPP and GGPP but not of cholesterol is specifically targeted in AD. Since prenylation of small GTPases by FPP or GGPP is indispensable for their proper function we are proposing that these two isoprenoids are up-regulated in AD resulting in an over abundance of certain prenylated proteins which contributes to neuronal dysfunction.

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®.

A rapid and sensitive assay for determining human brain levels of farnesyl-(FPP) and geranylgeranylpyrophosphate (GGPP) and transferase activities using UHPLC-MS/MS.

The isoprenoids farnesyl-(FPP) and geranylgeranylpyrophosphate (FPP and GGPP) are two major lipid intermediates in the mevalonate pathway. They participate in post-translational modification of members of the superfamily of small guanosine triphosphatases (GTPases; Ras, Rab, Rac, etc.) via prenylation reactions. Due to the important role of these proteins in a number of cell processes, in particular cell growth, division, and differentiation, investigation of the involvement of isoprenoids in these processes is of great interest. In a previously published report, we described a fully validated assay for the quantitation of the two isoprenoids using a high-performance liquid chromatography (HPLC)-fluorescence detection (FLD) method. The current work expands on the previous method and enhances it greatly by using a much faster state-of-the-art ultrahigh-performance liquid chromatography (UHPLC) technique coupled to tandem mass spectrometry (MS/MS). The method exhibited a linear concentration range of 5-250 ng/mL for FPP and GGPP in human brain tissue; it was shown to be unaffected by ion suppression and provided results almost six times faster than the HPLC-FLD assay. Comparison of UHPLC-MS/MS and HPLC-FLD yielded excellent comparability of the two assays for both isoprenoids. Based on the UHPLC-MS/MS assay, a novel in vitro test system was implemented to study enzyme specificity for distinct amino acid CAAX motifs, which is potentially useful for investigating target interactions of new therapeutics for diseases involving pathological regulation of isoprenoids and/or small GTPases.

Regulation of the brain isoprenoids farnesyl- and geranylgeranylpyrophosphate is altered in male Alzheimer patients.

Post-translational modification of small GTPases by farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP) has generated much attention due to their potential contribution to cancer, cardiovascular and neurodegenerative diseases. Prenylated proteins have been identified in numerous cell functions and elevated levels of FPP and GGPP have been previously proposed to occur in Alzheimer disease (AD) but have never been quantified. In the present study, we determined if the mevalonate derived compounds FPP and GGPP are increased in brain grey and white matter of male AD patients as compared with control samples. This study demonstrates for the first time that FPP and GGPP levels are significantly elevated in human AD grey and white matter but not cholesterol, indicating a potentially disease-specific targeting of isoprenoid regulation independent of HMG-CoA-reductase. Further suggesting a selective disruption of FPP and GGPP homeostasis in AD, we show that inhibition of HMG-CoA reductase in vivo significantly reduced FPP, GGPP and cholesterol abundance in mice with the largest effect on the isoprenoids. A tentative conclusion is that if indeed regulation of FPP and GGPP is altered in AD brain such changes may stimulate protein prenylation and contribute to AD neuropathophysiology.

Isoprenoid quantitation in human brain tissue: a validated HPLC-fluorescence detection method for endogenous farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP).

Farnesyl- and geranylgeranylpyrophosphate (FPP and GGPP) are isoprenoid intermediates in the mevalonate pathway. They play a crucial role in cell survival, growth and differentiation due to their attachment (isoprenylation) to small GTPases (Ras, Rho, etc.). Isoprenoid formation seems to be tightly regulated within the mevalonate pathway and its perturbation has been linked to certain diseases (e.g., cancer, Alzheimer's disease), but tissue levels are unknown. It is therefore of the utmost importance to quantify these isoprenoids in diseased tissue or in tissue after drug administration. The current work describes an isolation procedure utilizing a combination of Extrelut(R) liquid/liquid and reversed-phase solid-phase extraction (SPE) for homogenized human frontal cortex tissue. In addition, after a careful validation of an HPLC-fluorescence method, this assay allowed the determination of nanomolar concentrations of endogenous FPP and GGPP levels (4.5 and 10.6 ng/mg protein, respectively) in human brain tissue. The method is selective, precise (<15% RSD), accurate (<15% relative error) and sensitive over a linear range of 10-400 ng/mL for FPP and 50-1000 ng/mL for GGPP according to the current FDA criteria for bioanalytical method validation. Overall, this new method introduces the ability to simultaneously quantify FPP and GGPP in human brain tissue, and is potentially applicable to several other tissues and species.

State-of-the-art dried blood spot analysis: an overview of recent advances and future trends.

Dried blood spots have become a popular method in a variety of micro blood-sampling techniques in the life sciences sector, consequently competing with the field of conventional, invasive blood sampling by venepuncture. Dried blood spots are widely applied in numerous bioanalytical assays and have gained a significant role in the screening of inherited metabolic diseases, in PK and PD modeling; in the treatment and diagnosis of infectious diseases; and in therapeutic drug monitoring. Recent technological developments such as automation, online extraction, mass spectrometric direct analysis and also conventional dried blood spot bioanalysis, as well as future developments in dried blood spot bioanalysis are highlighted and presented in this article.

Brain isoprenoids farnesyl pyrophosphate and geranylgeranyl pyrophosphate are increased in aged mice.

The mevalonate/isoprenoids/cholesterol pathway has a fundamental role in the brain. Increasing age could be associated with specific changes in mevalonate downstream products. Other than age differences in brain cholesterol and dolichol levels, there has been little if any evidence on the short-chain isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP), as well as downstream lipid products. The purpose of the present study was to determine whether brain levels of FPP, GGPP and sterol precursors and metabolites would be altered in aged mice (23 months) as compared to middle-aged mice (12 months) and young mice (3 months). FPP and GGPP levels were found to be significantly higher in brain homogenates of 23-months-old mice. The ratio of FPP to GGPP did not differ among the three age groups suggesting that increasing age does not alter the relative distribution of the two isoprenoids. Gene expression of FPP synthase and GGPP synthase did not differ among the three age groups. Gene expression of HMG-CoA reductase was significantly increased with age but in contrast gene expression of squalene synthase was reduced with increasing age. Levels of squalene, lanosterol and lathosterol did not differ among the three age groups. Desmosterol and 7-dehydroxycholesterol, which are direct precursors in the final step of cholesterol biosynthesis were significantly lower in brains of aged mice. Levels of cholesterol and its metabolites 24S- and 25S-hydroxycholesterol were similar in all three age groups. Our novel find ings on increased FPP and GGPP levels in brains of aged mice may impact on protein prenylation and contribute to neuronal dysfunction observed in aging and certain neurodegenerative diseases.

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.

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.

Modulation of cholesterol, farnesylpyrophosphate, and geranylgeranylpyrophosphate in neuroblastoma SH-SY5Y-APP695 cells: impact on amyloid beta-protein production.

There is keen interest in the role of the isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) in protein prenylation and cell function in Alzheimer's disease (AD). We recently reported elevated FPP and GGPP brain levels and increased gene expression of FPP synthase (FPPS) and GGPP synthase (GGPPS) in the frontal cortex of AD patients. Cholesterol levels and gene expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were similar in AD and control samples, suggesting that homeostasis of FPP and GGPP but not cholesterol is specifically targeted in brain tissue of AD patients (Neurobiol Dis 2009 35:251-257). In the present study, it was determined if cellular levels of FPP, GGPP, and cholesterol affect beta-amyloid (Abeta) abundance in SH-SY5Y cells, expressing human APP695. Cells were treated with different inhibitors of the mevalonate/isoprenoid/cholesterol pathway. FPP, GGPP, cholesterol, and Abeta(1-40) levels were determined, and activities of farnesyltransferase and geranylgeranyltransferase I were measured. Inhibitors of different branches of the mevalonate/isoprenoid/cholesterol pathway as expected reduced cholesterol and isoprenoid levels in neuroblastoma cells. Abeta(1-40) levels were selectively reduced by cholesterol synthesis inhibitors but not by inhibitors of protein isoprenylation, indicating that changes in cholesterol levels per se and not isoprenoid levels account for the observed modifications in Abeta production.