Kersti Karu

Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols

In humans, the brain accounts for about 20% of the bodys free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MSn) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MSn (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7α,25-, and 7α,27-dihydroxycholesterols. In addition, 3β-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Induction of the Cytoprotective Enzyme Heme Oxygenase-1 by Statins Is Enhanced in Vascular Endothelium Exposed to Laminar Shear Stress and Impaired by Disturbed Flow

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H2O2-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.

Brain endogenous liver X receptor ligands selectively promote midbrain neurogenesis

Liver X receptors (Lxrα and Lxrβ) are ligand-dependent nuclear receptors critical for ventral midbrain neurogenesis in vivo. However, no endogenous midbrain Lxr ligand has so far been identified. Here we used LC/MS and functional assays to identify cholic acid as a new Lxr ligand. Moreover, 24(S),25-epoxycholesterol (24,25-EC) was found to be the most potent and abundant Lxr ligand in the developing mouse midbrain. Both Lxr ligands promoted neural development in an Lxr-dependent manner in zebrafish in vivo. Notably, each ligand selectively regulated the development of distinct midbrain neuronal populations. Whereas cholic acid increased survival and neurogenesis of Brn3a-positive red nucleus neurons, 24,25-EC promoted dopaminergic neurogenesis. These results identify an entirely new class of highly selective and cell type-specific regulators of neurogenesis and neuronal survival. Moreover, 24,25-EC promoted dopaminergic differentiation of embryonic stem cells, suggesting that Lxr ligands may thus contribute to the development of cell replacement and regenerative therapies for Parkinsons disease.

Nano-liquid chromatography–tandem mass spectrometry analysis of oxysterols in brain: monitoring of cholesterol autoxidation

Oxysterols are present in mammalian brain at ng/g-μg/g levels while cholesterol is present at the mg/g level. This makes oxysterol analysis of brain challenging. In an effort to meet this challenge we have developed, and validated, an isolation method based on solid phase extraction and an analytical protocol involving oxidation/derivatisation (i.e., charge-tagging) followed by nano-flow liquid chromatography (nano-LC) combined with tandem mass spectrometry utilising multi-stage fragmentation (MS(n)). The oxidation/derivatisation method employed improves detection limits by two orders of magnitude, while nano-LC-MS(n) provides separation of isomers and allows oxysterol quantification. Using this method 13 different oxysterols have been identified in rat brain including 24S-hydroxycholesterol, 24S,25-epoxycholesterol and 7α,26-dihydroxycholest-4-en-3-one. The level of 24S-hydroxycholesterol in rat brain was determined to be 20.3±3.4 μg/g and quantitative estimates were made for the other oxysterols identified. The presence of a large excess of cholesterol over oxysterol in brain raises the problem of autoxidation during sterol isolation and sample preparation. Thus, in parallel to identification studies, the degree of cholesterol autoxidation occurring during sterol isolation and analysis has been evaluated with the aid of [(2)H(7)]-labelled cholesterol and cholesterol autoxidation products identified.

24S,25-Epoxycholesterol in mouse and rat brain

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Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH2 groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.

Influence of PPCPs on the performance of intermittently operated slow sand filters for household water purification.

Removal of pharmaceuticals and personal care products (PPCPs) from drinking water is usually enhanced by advanced oxidation which is not affordable in low income countries. Slow sand filtration has been found to be capable of removing anti-inflammatory compounds, and its low maintenance costs and easy operation make it an attractive technology for treating drinking water in many parts of the world. In addition, slow sand filters can be used at both large and household scales. The biofilm (i.e. schmutzdecke) developed on the top of the sand and within the upper layers of the sand is acknowledged to be responsible for the water purification. However, it is possible that the PPCPs may affect the schmutzdecke development and microbial community within the filters, and consequently the performance of the filter. This study investigated two household slow sand filters (for water purification) operated intermittently with and without contamination by six PPCPs. Eleven parameters were monitored in the affluent and effluent water, including bacterial species present and schmutzdecke biomass development. Results demonstrated that the household slow sand filter performance was not affected by the 2μgL-1 of PPCPs in the water. There was no significant difference between filters for total coliforms and E. coli removal, but there was considerable difference between sampling times. Biomass considerably increased with the number of filtrations in both filters and there was no significant difference between filter biomass. However, it was found that more bacterial species were present in the period with no contamination than during the contamination period. Bacillus anthracis and Exiguobacterium sp. showed to be resistant to the effects of the PPCPs. These suggest there are effects of PPCPs on bacterial species within the filter. However, the effect of the PPCPs on biomass was not conclusive in this study and needs to be further investigated.

A highly sensitive liquid chromatography electrospray ionization mass spectrometry method for quantification of TMA, TMAO and creatinine in mouse urine

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Relative quantification of polyethylene glycol 400 excreted in the urine of male and female volunteers by direct injection electrospray-selected ion monitoring mass spectrometry

The use of polyethylene glycol 400 (PEG 400) as an excipient in oral formulations can have profound and differing effects on drug bioavailability in men and women; therefore an understanding of the pharmacokinetics of this excipient is required. A direct injection electrospray selected ion monitoring mass spectrometry methodology was developed and validated for the quantitation of PEG 400 excreted in human urine after oral administration. The most abundant ions corresponding to PEG 400 oligomers at m/z 365, 409, 453, 497, 541, and 585 were used for selected ion monitoring (SIM). Pre-dose urine of volunteers was spiked with various amounts of PEG 400 to generate calibration curves over the concentration range 2.5-90 μg/mL for all SIM channels. The relative standard deviations of intra- and inter-day analysis of PEG 400 in human urine were lower than 11.8% and bias percentage was less than 9.7%. This specific method for relative quantitation of PEG 400 was then used to analyse urine samples with minimal sample preparation. Urine samples of twelve healthy volunteers (six men and six women) who received 0.75 g and 1.5 g PEG 400 on two separate occasions were collected over 24h. On average 36.5% of the orally administered dose of PEG 400 was recovered in the urine of the volunteers, with no significant difference observed between men and women.

Analysis of transferred fragrance and its forensic implications

Perfumes are widely used by many people in developed countries, and a large number of both men and women wear perfumes on a daily basis. Analysis of perfume trace materials from clothing is not commonly employed within forensic casework, yet as a form of trace evidence it has the potential to provide valuable intelligence. In order to appreciate the value of trace evidence there is a fundamental need for an evidence base that can both offer insight into how a trace material behaves under different scenarios and activities, and from which inferences can be made. With this purpose a gas chromatography-mass spectrometry method for trace analysis of perfumes was developed. This paper presents two different series of experiments that investigate the dynamics of perfume transfer as a factor of perfume ageing time, and as a factor of perfume contact time. Empirical data showed that both perfume ageing time, and perfume contact time play a key role in the number of perfume components transferred. These studies have implication for forensic protocols, specifically for perfume trace evidence collection, analysis, interpretation, and presentation, and there is potentially great value in analysing perfumes from clothing exhibits in forensic enquiries that involve close contact between individuals, such as sexual assaults.