Cholestasis impairs hepatic lipid storage via AMPK and CREB signaling in hepatitis B virus surface protein transgenic mice.

Clinical studies demonstrated that nonalcoholic steatohepatitis is associated with liver-related outcomes in chronic hepatitis B. Furthermore, primary biliary fibrosis and biliary atresia occurred in patients with HBV infection. Interestingly, hepatitis B virus surface protein (HBs) transgenic mice spontaneously develop hepatic steatosis. Our aim is to investigate the effect of Abcb4 knockout-induced cholestasis on liver steatosis in HBs transgenic mice. Hybrids of HBs transgenic and Abcb4-/- mice were bred on the BALB/c genetic background. Lipid synthesis, storage, and catabolism as well as proteins and genes that control lipid metabolism were analyzed using HPTLC, qPCR, western blot, electrophoretic mobility shift assay (EMSA), lipid staining, and immunohistochemistry. Hepatic neutral lipid depots were increased in HBs transgenic mice and remarkably reduced in Abcb4-/- and HBs/Abcb4-/- mice. Similarly, HPTLC-based quantification analyses of total hepatic lipid extracts revealed a significant reduction in the amount of triacylglycerols (TAG), while the amount of free fatty acids (FFA) was increased in Abcb4-/- and HBs/Abcb4-/- in comparison to wild-type and HBs mice. PLIN2, a lipid droplet-associated protein, was less expressed in Abcb4-/- and HBs/Abcb4-/-. The expression of genes-encoding proteins involved in TAG synthesis and de novo lipogenesis (Agpat1, Gpat1, Mgat1, Dgat1, Dgat2, Fasn, Hmgcs1, Acc1, Srebp1-c, and Pparγ) was suppressed, and AMPK and CREB were activated in Abcb4-/- and HBs/Abcb4-/- compared to wild-type and HBs mice. Simulating cholestatic conditions in cell culture resulted in AMPK and CREB activation while FASN and PLIN2 were reduced. A concurrent inhibition of AMPK signaling revealed normal expression level of FASN and PLIN2, suggesting that activation of AMPK-CREB signaling regulates hepatic lipid metabolism, i.e. synthesis and storage, under cholestatic condition. In conclusions, in vivo and mechanistic in vitro data suggest that cholestasis reduces hepatic lipid storage via AMPK and CREB signaling. The results of the current study could be the basis for novel therapeutic strategies as NASH is a crucial factor that can aggravate chronic liver diseases.

Nanomole-scaled high-throughput chemistry plus direct bioautography on the same chromatography plate for drug discovery.

The powerful fusion of on-surface synthesis and effect-directed analysis was introduced as novel tool for synthetic drug discovery, all on the same high-performance thin-layer chromatography plate. Precise automated sample application allowed both, high-throughput chemistry of 60 reactions at once and reaction miniaturization down to the 15-nmol scale. The antibiotic activity of all on-surface synthesized compounds was evaluated on the same surface via the Gram-positive Bacillus subtilis bioassay. For one product, synthesis (reaction, purification and identification) took 5.3 min and semi-quantitative biological evaluation took 2.8 min. Out of 60 on-surface reactions 10 products (17%) were identified to be more active than a well-known antibiotic reference. The concept was transferred to the Gram-negative Aliivibrio fischeri bioassay. For the first time, a new analytical platform was shown for a streamlined workflow at the most miniaturized scale from synthesis, purification, identification and quantification to semi-quantitative biological activity evaluation (all on the same chromatography plate).

Quantitative inkjet application on self-printed, binder-free HPTLC layers for submicromole-scaled analytical 1H NMR spectroscopy.

High-performance thin-layer chromatography (HPTLC) combined with 1H nuclear magnetic resonance (NMR) spectroscopy has only been demonstrated over a small spectral range so far. The self-printing of chromatographic plates with a modified 3D slurry printer allowed the operator to influence the composition of the adsorbent slurry and thus the purity of the adsorbent layer. The combination of such self-printed, binder-free HPTLC plates with inkjet-driven sample application made possible submicromole-scaled analytical 1H NMR spectroscopy. This was proven using pure HPTLC adsorbents. For comparison, commercial silica gel HPTLC plates were purified by pre-development with solvents for spectroscopy, whereas commercial silica gel HPTLC particles were self-printed on the glass plate after purification under solvent pressure in a recycled HPLC cartridge. Evaluating the signals from different treatments, seven background signals disappeared in the proton spectra and three were reduced to a minimum by use of pre-developed commercial HPTLC plates. In the case of the self-printed, binder-free HPTLC plates made of purified adsorbent, most of the spectral background signals were reduced to a minimum, thus these spectra showed the highest cleanness and most pure analyte proton spectra. For the first time, the full 1H NMR spectroscopy range was made available after an HPTLC separation. This proof of principle opens the avenue for submicromole-scaled analytical 1H NMR spectroscopy.

eicCluster software, an open-source in silico tool, and on-surface syntheses, an in situ concept, both exploited for signal highlighting in high-resolution mass spectrometry to ease structure elucidation in planar chromatography.

Optimization of the ionization parameters in mass spectrometry does not guarantee a sufficient response and successful signal assignment when analyzing unknown compounds presenting signals of low intensity. The interpretation is difficult as the mass signals of degradation products are often less intense than background signals. Important information may be overlooked. Such critical and time consuming data analysis was overcome by developing a new strategy and open-source software called eicCluster offering unsupervised machine learning algorithms and powerful interactive visualization tools that made data processing fast and intuitive. Using eicCluster for high-performance thin-layer chromatography coupled with high-resolution mass spectrometry, mass signals of degradation products were highlighted in a stressed formulation, which were hardly found until linked to the new software. Owed to (1) preprocessing leading to intensity-agnostic signals and (2) the t-SNE algorithm, clustering mass signals based on their similarity, even compound ions present at low intensities were separated in subclusters from background signals (in silico highlighting). The resulting 2D maps allowed a new view on the data set to target molecules (degradation products) in complex mixtures. In addition to this new source of information, targeted on-surface synthesis of degradation products (in situ highlighting) was shown to support a fast structure elucidation when standards are not commercially available. It allowed a better understanding of the proposed degradation reactions in the formulation. As proof of principle, an original example formulation, its stressed samples as well as the proposed degradation products of on-surface synthesis were compared. In silico and in situ signal highlighting substantially eased data processing in structure elucidation.

All on one high-performance thin-layer chromatography plate: solvent-free nanomole-scaled on-surface synthesis, workup and online high-resolution mass spectrometry for elucidation of two new degradation products in an ifosfamide formulation.

On-surface reactions were introduced as a new strategy for rapid structure elucidation. This strategy is illustrated by the miniaturized synthesis-guided identification of two new degradation products (impurities) occurring in a pharmaceutical formulation of the anti-cancer drug ifosfamide, especially in the presence of urea. Synthesis on the silica gel surface bypassed the need for solvents, as the large nm-porous surface favoured a fast conversion of the reaction partners. For the on-surface synthesis of the impurities, the respective reagents were accurately and automatedly applied in the nanomole scale on a high-performance thin-layer chromatography (HPTLC) silica gel plate. After a fast reaction, the workup of the reaction mixture was performed by development of the HPTLC plate followed by online high-resolution mass spectrometry. As proof of concept and for benchmarking, a reaction mixture obtained from conventional preparative synthesis in a round-bottom flask was analysed in parallel as well as different formulations. The use of adsorbents as inert layer turned out to be highly efficient for a rapid generation and confirmation of impurities, as the synthesis on the HPTLC layer revealed them within 10 min. Image evaluation was simply performed by videodensitometry. The advantageous combination of all steps on one HPTLC plate and its resulting efficiency made surface synthesis on chromatographic phases an optimal tool for signal highlighting in mass spectrometry, and thus for the assignment of impurities in drugs. The chemistry process scale was miniaturized down to the µg-level per synthesis (in total 30-60 µg chemicals/reaction), setting a new state-of-the-art standard. All material savings clearly contribute to green chemistry, and this strategy substantially reduces the consumption of chemicals and greatly enhances the analytical efficiency, when adapted by scientists for the quality control of any other chemical product. The combination of synthesis, workup and detection in a miniaturized process, contributes to optimized workflows in a lean laboratory.

Effect-directed analysis via hyphenated high-performance thin-layer chromatography for bioanalytical profiling of sunflower leaves.

High-performance thin-layer chromatography (HPTLC) coupled with effect-directed analysis was used for non-targeted screening of sunflower leaf extract for components exhibiting antioxidant, antibacterial and/or cholinesterase enzyme inhibitory effects. The active compounds were characterized by HPTLC-electrospray ionization-high resolution mass spectrometry (ESI-HRMS) and HPTLC-Direct Analysis in Real Time (DART)-MS/MS. The latter ambient ionization technique (less soft than ESI) resulted in oxidation and fragmentation products and characteristic fragment ions. NMR spectroscopy after targeted isolation via preparative normal phase flash chromatography and semi-preparative reversed phase high-performance liquid chromatography supported the identification of two diterpenes to be (-)-kaur-16-en-19-oic acid and 15-α-angeloyloxy-ent-kaur-16-en-19-oic acid. Both compounds found to be multi-potent as they inhibited acetylcholinesterase and butyrylcholinesterase and showed antibacterial effects against Gram-positive Bacillus subtilis and Gram-negative Aliivibrio fischeri bacteria. Kaurenoic acid was also active against the Gram-negative pepper pathogenic Xanthomonas euvesicatoria bacteria.

Ultrafast dynamics of differently aligned COOH-DTE-BODIPY conjugates linked to the surface of TiO2.

The photoinduced dynamics of two DTE-BODIPY conjugates A, B with carboxylic acid anchoring groups coupled to the surface of TiO2 were studied by ultrafast transient absorption spectroscopy. For compound A, with an orthogonal orientation of the BODIPY chromophore and the photoswitchable DTE unit, a charge separated state could not be reliably detected. Nevertheless, besides the energy transfer from the BODIPY to the ring-closed DTE-c, indications for an electron transfer reaction were found by analyzing fluorescence quenching on TiO2 in steady state fluorescence measurements. For compound B with a parallel orientation of chromophore and photoswitch, a charge separated state was conclusively identified for the coupled dyad (TiO2) via the observation of a positive absorption signal (at λ pr > 610 nm) at later delay times. An electron transfer rate of 7 × 1010 s-1 can be extracted, indicating slower processes in the dyads in comparison to previously published electron transfer reactions of DTE compounds coupled to TiO2.

Streamlined structure elucidation of an unknown compound in a pigment formulation.

A fast and reliable quality control is important for ink manufacturers to ensure a constant production grade of mixtures and chemical formulations, and unknown components attract their attention. Structure elucidating techniques seem time-consuming in combination with column-based methods, but especially the low solubility of pigment formulations is challenging the analysis. In contrast, layer chromatography is more tolerant with regard to pigment particles. One PLC plate for NMR and FTIR analyses and one HPTLC plate for recording of high resolution mass spectra, MS/MS spectra and for gathering information on polarity and spectral properties were needed to characterize a structure, exemplarily shown for an unknown component in pigment Red 57:1 to be 3-hydroxy-2-naphtoic acid. A preparative layer chromatography (PLC) workflow was developed that used an Automated Multiple Development 2 (AMD 2) system. The 0.5-mm PLC plate could still be operated in the AMD 2 system and allowed a smooth switch from the analytical to the preparative gradient separation. Through automated gradient development and the resulting focusing of bands, the sharpness of the PLC bands was improved. For NMR, the necessary high load of the target compound on the PLC plate was achieved via a selective solvent extraction that discriminated the polar sample matrix and thus increased the application volume of the extract that could maximally be applied without overloading. By doing so, the yield for NMR analysis was improved by a factor of 9. The effectivity gain through a simple, but thoroughly chosen extraction solvent is often overlooked, and for educational purpose, it was clearly illustrated and demonstrated by an extended solvent screening. Thus, PLC using an automated gradient development after a selective extraction was proven to be a new powerful combination for structural elucidation by NMR.