Liquid Chromatography-Mass Spectrometry in Studies of Drug Metabolism and Permeability

Poor pharmacokinetics is one of the reasons for the withdrawal of drug candidates from clinical trials. There is an urgent need for investigating in vitro ADME (absorption, distribution, metabolism and excretion) properties and recognising unsuitable drug candidates as early as possible in the drug development process. Current throughput of in vitro ADME profiling is insufficient because effective new synthesis techniques, such as drug design in silico and combinatorial synthesis, have vastly increased the number of drug candidates. Assay technologies for larger sets of compounds than are currently feasible are critically needed. The first part of this work focused on the evaluation of cocktail strategy in studies of drug permeability and metabolic stability. N-in-one liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods were developed and validated for the multiple component analysis of samples in cocktail experiments. Together, cocktail dosing and LC/MS/MS were found to form an effective tool for increasing throughput. First, cocktail dosing, i.e. the use of a mixture of many test compounds, was applied in permeability experiments with Caco-2 cell culture, which is a widely used in vitro model for small intestinal absorption. A cocktail of 7-10 reference compounds was successfully evaluated for standardization and routine testing of the performance of Caco-2 cell cultures. Secondly, cocktail strategy was used in metabolic stability studies of drugs with UGT isoenzymes, which are one of the most important phase II drug metabolizing enzymes. The study confirmed that the determination of intrinsic clearance (Clint) as a cocktail of seven substrates is possible. The LC/MS/MS methods that were developed were fast and reliable for the quantitative analysis of a heterogenous set of drugs from Caco-2 permeability experiments and the set of glucuronides from in vitro stability experiments. The performance of a new ionization technique, atmospheric pressure photoionization (APPI), was evaluated through comparison with electrospray ionization (ESI), where both techniques were used for the analysis of Caco-2 samples. Like ESI, also APPI proved to be a reliable technique for the analysis of Caco-2 samples and even more flexible than ESI because of the wider dynamic linear range. The second part of the experimental study focused on metabolite profiling. Different mass spectrometric instruments and commercially available software tools were investigated for profiling metabolites in urine and hepatocyte samples. All the instruments tested (triple quadrupole, quadrupole time-of-flight, ion trap) exhibited some good and some bad features in searching for and identifying of expected and non-expected metabolites. Although, current profiling software is helpful, it is still insufficient. Thus a time-consuming largely manual approach is still required for metabolite profiling from complex biological matrices.

Accurate mass-based analysis in human sport doping control : Application of liquid chromatography - time-of-flight mass spectrometry

Human sport doping control analysis is a complex and challenging task for anti-doping laboratories. The List of Prohibited Substances and Methods, updated annually by World Anti-Doping Agency (WADA), consists of hundreds of chemically and pharmacologically different low and high molecular weight compounds. This poses a considerable challenge for laboratories to analyze for them all in a limited amount of time from a limited sample aliquot. The continuous expansion of the Prohibited List obliges laboratories to keep their analytical methods updated and to research new available methodologies. In this thesis, an accurate mass-based analysis employing liquid chromatography - time-of-flight mass spectrometry (LC-TOFMS) was developed and validated to improve the power of doping control analysis. New analytical methods were developed utilizing the high mass accuracy and high information content obtained by TOFMS to generate comprehensive and generic screening procedures. The suitability of LC-TOFMS for comprehensive screening was demonstrated for the first time in the field with mass accuracies better than 1 mDa. Further attention was given to generic sample preparation, an essential part of screening analysis, to rationalize the whole work flow and minimize the need for several separate sample preparation methods. Utilizing both positive and negative ionization allowed the detection of almost 200 prohibited substances. Automatic data processing produced a Microsoft Excel based report highlighting the entries fulfilling the criteria of the reverse data base search (retention time (RT), mass accuracy, isotope match). The quantitative performance of LC-TOFMS was demonstrated with morphine, codeine and their intact glucuronide conjugates. After a straightforward sample preparation the compounds were analyzed directly without the need for hydrolysis, solvent transfer, evaporation or reconstitution. The hydrophilic interaction technique (HILIC) provided good chromatographic separation, which was critical for the morphine glucuronide isomers. A wide linear range (50-5000 ng/ml) with good precision (RSD<10%) and accuracy (±10%) was obtained, showing comparable or better performance to other methods used. In-source collision-induced dissociation (ISCID) allowed confirmation analysis with three diagnostic ions with a median mass accuracy of 1.08 mDa and repeatable ion ratios fulfilling WADA s identification criteria. The suitability of LC-TOFMS for screening of high molecular weight doping agents was demonstrated with plasma volume expanders (PVE), namely dextran and hydroxyethylstarch (HES). Specificity of the assay was improved, since interfering matrix compounds were removed by size exclusion chromatography (SEC). ISCID produced three characteristic ions with an excellent mean mass accuracy of 0.82 mDa at physiological concentration levels. In summary, by combining TOFMS with a proper sample preparation and chromatographic separation, the technique can be utilized extensively in doping control laboratories for comprehensive screening of chemically different low and high molecular weight compounds, for quantification of threshold substances and even for confirmation. LC-TOFMS rationalized the work flow in doping control laboratories by simplifying the screening scheme, expediting reporting and minimizing the analysis costs. Therefore LC-TOFMS can be exploited widely in doping control, and the need for several separate analysis techniques is reduced.

Diastereomeric Drug Glucuronides: Enzymatic Glucuronidation and Analysis by Capillary Electrophoresis and Liquid Chromatography-Mass Spectrometry

Foreign compounds, such as drugs are metabolised in the body in numerous reactions. Metabolic reactions are divided into phase I (functionalisation) and phase II (conjugation) reactions. Uridine diphosphoglucuronosyltransferase enzymes (UGTs) are important catalysts of phase II metabolic system. They catalyse the transfer of glucuronic acid to small lipophilic molecules and convert them to hydrophilic and polar glucuronides that are readily excreted from the body. Liver is the main site of drug metabolism. Many drugs are racemic mixtures of two enantiomers. Glucuronidation of a racemic compound yields a pair of diastereomeric glucuronides. Stereoisomers are interesting substrates in glucuronidation studies since some UGTs display stereoselectivity. Diastereomeric glucuronides of O-desmethyltramadol (M1) and entacapone were selected as model compounds in this work. The investigations of the thesis deal with enzymatic glucuronidation and the development of analytical methods for drug metabolites, particularly diastereomeric glucuronides. The glucuronides were analysed from complex biological matrices, such as urine or from in vitro incubation matrices. Various pretreatment techniques were needed to purify, concentrate and isolate the analytes of interest. Analyses were carried out by liquid chromatography (LC) with ultraviolet (UV) or mass spectrometric (MS) detection or with capillary electromigration techniques. Commercial glucuronide standards were not available for the studies. Enzyme-assisted synthesis with rat liver microsomes was therefore used to produce M1 glucuronides as reference compounds. The glucuronides were isolated by LC/UV and ultra performance liquid chromatography (UPLC)/MS, while tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) spectroscopy were employed in structural characterisation. The glucuronides were identified as phenolic O-glucuronides of M1. To identify the active UGT enzymes in (±)-M1 glucuronidation recombinant human UGTs and human tissue microsomes were incubated with (±)-M1. The study revealed that several UGTs can catalyse (±)-M1 glucuronidation. Glucuronidation in human liver microsomes like in rat liver microsomes is stereoselective. The results of the studies showed that UGT2B7, most probably, is the main UGT responsible for (±)-M1 glucuronidation in human liver. Large variation in stereoselectivity of UGTs toward (±)-M1 enantiomers was observed. Formation of M1 glucuronides was monitored with a fast and selective UPLC/MS method. Capillary electromigration techniques are known for their high resolution power. A method that relied on capillary electrophoresis (CE) with UV detection was developed for the separation of tramadol and its free and glucuronidated metabolites. The suitability of the method to identify tramadol metabolites in an authentic urine samples was tested. Unaltered tramadol and four of its main metabolites were detected in the electropherogram. A micellar electrokinetic chromatography (MEKC) /UV method was developed for the separation of the glucuronides of entacapone in human urine. The validated method was tested in the analysis of urine samples of patients. The glucuronides of entacapone could be quantified after oral entacapone dosing.

Development of Sample Pretreatment and Liquid Chromatographic Techniques for Antioxidative Compounds

In this study, novel methodologies for the determination of antioxidative compounds in herbs and beverages were developed. Antioxidants are compounds that can reduce, delay or inhibit oxidative events. They are a part of the human defense system and are obtained through the diet. Antioxidants are naturally present in several types of foods, e.g. in fruits, beverages, vegetables and herbs. Antioxidants can also be added to foods during manufacturing to suppress lipid oxidation and formation of free radicals under conditions of cooking or storage and to reduce the concentration of free radicals in vivo after food ingestion. There is growing interest in natural antioxidants, and effective compounds have already been identified from antioxidant classes such as carotenoids, essential oils, flavonoids and phenolic acids. The wide variety of sample matrices and analytes presents quite a challenge for the development of analytical techniques. Growing demands have been placed on sample pretreatment. In this study, three novel extraction techniques, namely supercritical fluid extraction (SFE), pressurised hot water extraction (PHWE) and dynamic sonication-assisted extraction (DSAE) were studied. SFE was used for the extraction of lycopene from tomato skins and PHWE was used in the extraction of phenolic compounds from sage. DSAE was applied to the extraction of phenolic acids from Lamiaceae herbs. In the development of extraction methodologies, the main parameters of the extraction were studied and the recoveries were compared to those achieved by conventional extraction techniques. In addition, the stability of lycopene was also followed under different storage conditions. For the separation of the antioxidative compounds in the extracts, liquid chromatographic methods (LC) were utilised. Two novel LC techniques, namely ultra performance liquid chromatography (UPLC) and comprehensive two-dimensional liquid chromatography (LCxLC) were studied and compared with conventional high performance liquid chromatography (HPLC) for the separation of antioxidants in beverages and Lamiaceae herbs. In LCxLC, the selection of LC mode, column dimensions and flow rates were studied and optimised to obtain efficient separation of the target compounds. In addition, the separation powers of HPLC, UPLC, HPLCxHPLC and HPLCxUPLC were compared. To exploit the benefits of an integrated system, in which sample preparation and final separation are performed in a closed unit, dynamic sonication-assisted extraction was coupled on-line to a liquid chromatograph via a solid-phase trap. The increased sensitivity was utilised in the extraction of phenolic acids from Lamiaceae herbs. The results were compared to those of achieved by the LCxLC system.

Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation

This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of microchannel, which leads to a sharp electrospray tip. Microchannel contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on-chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry.

Heated Nebulizer Microchips for Mass Spectrometry

Miniaturized analytical devices, such as heated nebulizer (HN) microchips studied in this work, are of increasing interest owing to benefits like faster operation, better performance, and lower cost relative to conventional systems. HN microchips are microfabricated devices that vaporize liquid and mix it with gas. They are used with low liquid flow rates, typically a few µL/min, and have previously been utilized as ion sources for mass spectrometry (MS). Conventional ion sources are seldom feasible at such low flow rates. In this work HN chips were developed further and new applications were introduced. First, a new method for thermal and fluidic characterization of the HN microchips was developed and used to study the chips. Thermal behavior of the chips was also studied by temperature measurements and infrared imaging. An HN chip was applied to the analysis of crude oil – an extremely complex sample – by microchip atmospheric pressure photoionization (APPI) high resolution mass spectrometry. With the chip, the sample flow rate could be reduced significantly without loss of performance and with greatly reduced contamination of the MS instrument. Thanks to its suitability to high temperature, microchip APPI provided efficient vaporization of nonvolatile compounds in crude oil. The first microchip version of sonic spray ionization (SSI) was presented. Ionization was achieved by applying only high (sonic) speed nebulizer gas to an HN microchip. SSI significantly broadens the range of analytes ionizable with the HN chips, from small stable molecules to labile biomolecules. The analytical performance of the microchip SSI source was confirmed to be acceptable. The HN microchips were also used to connect gas chromatography (GC) and capillary liquid chromatography (LC) to MS, using APPI for ionization. Microchip APPI allows efficient ionization of both polar and nonpolar compounds whereas with the most popular electrospray ionization (ESI) only polar and ionic molecules are ionized efficiently. The combination of GC with MS showed that, with HN microchips, GCs can easily be used with MS instruments designed for LC-MS. The presented analytical methods showed good performance. The first integrated LC–HN microchip was developed and presented. In a single microdevice, there were structures for a packed LC column and a heated nebulizer. Nonpolar and polar analytes were efficiently ionized by APPI. Ionization of nonpolar and polar analytes is not possible with previously presented chips for LC–MS since they rely on ESI. Preliminary quantitative performance of the new chip was evaluated and the chip was also demonstrated with optical detection. A new ambient ionization technique for mass spectrometry, desorption atmospheric pressure photoionization (DAPPI), was presented. The DAPPI technique is based on an HN microchip providing desorption of analytes from a surface. Photons from a photoionization lamp ionize the analytes via gas-phase chemical reactions, and the ions are directed into an MS. Rapid analysis of pharmaceuticals from tablets was successfully demonstrated as an application of DAPPI.

Thin-Layer Chromatography with Ultraviolet and Mass Spectrometric Detection: From Preparative-Layer to Miniaturized Ultra-Thin-Layer Technique

The present challenge in drug discovery is to synthesize new compounds efficiently in minimal time. The trend is towards carefully designed and well-characterized compound libraries because fast and effective synthesis methods easily produce thousands of new compounds. The need for rapid and reliable analysis methods is increased at the same time. Quality assessment, including the identification and purity tests, is highly important since false (negative or positive) results, for instance in tests of biological activity or determination of early-ADME parameters in vitro (the pharmacokinetic study of drug absorption, distribution, metabolism, and excretion), must be avoided. This thesis summarizes the principles of classical planar chromatographic separation combined with ultraviolet (UV) and mass spectrometric (MS) detection, and introduces powerful, rapid, easy, low-cost, and alternative tools and techniques for qualitative and quantitative analysis of small drug or drug-like molecules. High performance thin-layer chromatography (HPTLC) was introduced and evaluated for fast semi-quantitative assessment of the purity of synthesis target compounds. HPTLC methods were compared with the liquid chromatography (LC) methods. Electrospray ionization mass spectrometry (ESI MS) and atmospheric pressure matrix-assisted laser desorption/ionization MS (AP MALDI MS) were used to identify and confirm the product zones on the plate. AP MALDI MS was rapid, and easy to carry out directly on the plate without scraping. The PLC method was used to isolate target compounds from crude synthesized products and purify them for bioactivity and preliminary ADME tests. Ultra-thin-layer chromatography (UTLC) with AP MALDI MS and desorption electrospray ionization mass spectrometry (DESI MS) was introduced and studied for the first time. Because of the thinner adsorbent layer, the monolithic UTLC plate provided 10 100 times better sensitivity in MALDI analysis than did HPTLC plates. The limits of detection (LODs) down to low picomole range were demonstrated for UTLC AP MALDI and UTLC DESI MS. In a comparison of AP and vacuum MALDI MS detection for UTLC plates, desorption from the irregular surface of the plates with the combination of an external AP MALDI ion source and an ion trap instrument provided clearly less variation in mass accuracy than the vacuum MALDI time-of-flight (TOF) instrument. The performance of the two-dimensional (2D) UTLC separation with AP MALDI MS method was studied for the first time. The influence of the urine matrix on the separation and the repeatability was evaluated with benzodiazepines as model substances in human urine. The applicability of 2D UTLC AP MALDI MS was demonstrated in the detection of metabolites in an authentic urine sample.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Comprehensive Two-Dimensional Gas Chromatography: Instrumental and Methodological Development

Comprehensive two-dimensional gas chromatography (GC×GC) offers enhanced separation efficiency, reliability in qualitative and quantitative analysis, capability to detect low quantities, and information on the whole sample and its components. These features are essential in the analysis of complex samples, in which the number of compounds may be large or the analytes of interest are present at trace level. This study involved the development of instrumentation, data analysis programs and methodologies for GC×GC and their application in studies on qualitative and quantitative aspects of GC×GC analysis. Environmental samples were used as model samples. Instrumental development comprised the construction of three versions of a semi-rotating cryogenic modulator in which modulation was based on two-step cryogenic trapping with continuously flowing carbon dioxide as coolant. Two-step trapping was achieved by rotating the nozzle spraying the carbon dioxide with a motor. The fastest rotation and highest modulation frequency were achieved with a permanent magnetic motor, and modulation was most accurate when the motor was controlled with a microcontroller containing a quartz crystal. Heated wire resistors were unnecessary for the desorption step when liquid carbon dioxide was used as coolant. With use of the modulators developed in this study, the narrowest peaks were 75 ms at base. Three data analysis programs were developed allowing basic, comparison and identification operations. Basic operations enabled the visualisation of two-dimensional plots and the determination of retention times, peak heights and volumes. The overlaying feature in the comparison program allowed easy comparison of 2D plots. An automated identification procedure based on mass spectra and retention parameters allowed the qualitative analysis of data obtained by GC×GC and time-of-flight mass spectrometry. In the methodological development, sample preparation (extraction and clean-up) and GC×GC methods were developed for the analysis of atmospheric aerosol and sediment samples. Dynamic sonication assisted extraction was well suited for atmospheric aerosols collected on a filter. A clean-up procedure utilising normal phase liquid chromatography with ultra violet detection worked well in the removal of aliphatic hydrocarbons from a sediment extract. GC×GC with flame ionisation detection or quadrupole mass spectrometry provided good reliability in the qualitative analysis of target analytes. However, GC×GC with time-of-flight mass spectrometry was needed in the analysis of unknowns. The automated identification procedure that was developed was efficient in the analysis of large data files, but manual search and analyst knowledge are invaluable as well. Quantitative analysis was examined in terms of calibration procedures and the effect of matrix compounds on GC×GC separation. In addition to calibration in GC×GC with summed peak areas or peak volumes, simplified area calibration based on normal GC signal can be used to quantify compounds in samples analysed by GC×GC so long as certain qualitative and quantitative prerequisites are met. In a study of the effect of matrix compounds on GC×GC separation, it was shown that quality of the separation of PAHs is not significantly disturbed by the amount of matrix and quantitativeness suffers only slightly in the presence of matrix and when the amount of target compounds is low. The benefits of GC×GC in the analysis of complex samples easily overcome some minor drawbacks of the technique. The developed instrumentation and methodologies performed well for environmental samples, but they could also be applied for other complex samples.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Sleep deprivation and brain energy metabolism : in vivo studies in rats and humans

Sleep deprivation leads to increased subsequent sleep length and depth and to deficits in cognitive performance in humans. In animals extreme sleep deprivation is eventually fatal. The cellular and molecular mechanisms causing the symptoms of sleep deprivation are unclear. This thesis was inspired by the hypothesis that during wakefulness brain energy stores would be depleted, and they would be replenished during sleep. The aim of this thesis was to elucidate the energy metabolic processes taking place in the brain during sleep deprivation. Endogenous brain energy metabolite levels were assessed in vivo in rats and in humans in four separate studies (Studies I-IV). In the first part (Study I) the effects of local energy depletion on brain energy metabolism and sleep were studied in rats with the use of in vivo microdialysis combined with high performance liquid chromatography. Energy depletion induced by 2,4-dinitrophenol infusion into the basal forebrain was comparable to the effects of sleep deprivation: both increased extracellular concentrations of adenosine, lactate, and pyruvate, and elevated subsequent sleep. This result supports the hypothesis of a connection between brain energy metabolism and sleep. The second part involved healthy human subjects (Studies II-IV). Study II aimed to assess the feasibility of applying proton magnetic resonance spectroscopy (1H MRS) to study brain lactate levels during cognitive stimulation. Cognitive stimulation induced an increase in lactate levels in the left inferior frontal gyrus, showing that metabolic imaging of neuronal activity related to cognition is possible with 1H MRS. Study III examined the effects of sleep deprivation and aging on the brain lactate response to cognitive stimulation. No physiologic, cognitive stimulation-induced lactate response appeared in the sleep-deprived and in the aging subjects, which can be interpreted as a sign of malfunctioning of brain energy metabolism. This malfunctioning may contribute to the functional impairment of the frontal cortex both during aging and sleep deprivation. Finally (Study IV), 1H MRS major metabolite levels in the occipital cortex were assessed during sleep deprivation and during photic stimulation. N-acetyl-aspartate (NAA/H2O) decreased during sleep deprivation, supporting the hypothesis of sleep deprivation-induced disturbance in brain energy metabolism. Choline containing compounds (Cho/H2O) decreased during sleep deprivation and recovered to alert levels during photic stimulation, pointing towards changes in membrane metabolism, and giving support to earlier observations of altered brain response to stimulation during sleep deprivation. Based on these findings, it can be concluded that sleep deprivation alters brain energy metabolism. However, the effects of sleep deprivation on brain energy metabolism may vary from one brain area to another. Although an effect of sleep deprivation might not in all cases be detectable in the non-stimulated baseline state, a challenge imposed by cognitive or photic stimulation can reveal significant changes. It can be hypothesized that brain energy metabolism during sleep deprivation is more vulnerable than in the alert state. Changes in brain energy metabolism may participate in the homeostatic regulation of sleep and contribute to the deficits in cognitive performance during sleep deprivation.

The role of catechol-O-methyltransferase (COMT) and the effects of COMT inhibition in brain and in cardiovascular and renal pathophysiology

Catechol-O-methyltransferase (COMT) metabolizes catecholamines such as dopamine (DA), noradrenaline (NA) and adrenaline, which are vital neurotransmitters and hormones that play important roles in the regulation of physiological processes. COMT enzyme has a functional Val158Met polymorphism in humans, which affects the subjects COMT activity. Increasing evidence suggests that this functional polymorphism may play a role in the etiology of various diseases from schizophrenia to cancers. The aim of this project was to provide novel biochemical information on the physiological and especially pathophysiological roles of COMT enzyme as well as the effects of COMT inhibition in the brain and in the cardiovascular and renal system. To assess the roles of COMT and COMT inhibition in pathophysiology, we used four different study designs. The possible beneficial effects of COMT inhibition were studied in double-transgenic rats (dTGRs) harbouring human angiotensinogen and renin genes. Due to angiotensin II (Ang II) overexpression, these animals exhibit severe hypetension, cardiovascular and renal end-organ damage and mortality of approximately 25-40% at the age of 7-weeks. The dTGRs and their Sprague-Dawley controls tissue samples were assessed with light microscopy, immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and high-pressure liquid chromatography (HPLC) to evaluate the tissue damages and the possible protective effects pharmacological intervention with COMT inhibitors. In a second study, the consequence of genetic and pharmacological COMT blockade in blood pressure regulation during normal and high-sodium was elucidated using COMT-deficient mice. The blood pressure and the heart rate were measured using direct radiotelemetric blood pressure surveillance. In a third study, the effects of acute and subchronic COMT inhibition during combined levodopa (L-DOPA) + dopa decarboxylase inhibitor treatment in homocysteine formation was evaluated. Finally, we assessed the COMT enzyme expression, activity and cellular localization in the CNS during inflammation-induced neurodegeneration using Western blotting, HPLC and various enzymatic assays. The effects of pharmacological COMT inhibition on neurodegeneration were also studied. The COMT inhibitor entacapone protected against the Ang II-induced perivascular inflammation, renal damage and cardiovascular mortality in dTGRs. COMT inhibitors reduced the albuminuria by 85% and prevented the cardiovascular mortality completely. Entacapone treatment was shown to ameliorate oxidative stress and inflammation. Furthermore, we established that the genetic and pharmacological COMT enzyme blockade protects against the blood pressure-elevating effects of high sodium intake in mice. These effects were mediated via enhanced renal dopaminergic tone and suggest an important role of COMT enzyme, especially in salt-sensitive hypertension. Entacapone also ameliorated the L-DOPA-induced hyperhomocysteinemia in rats. This is important, since decreased homocysteine levels may decrease the risk of cardiovascular diseases in Parkinson´s disease (PD) patients using L-DOPA. The Lipopolysaccharide (LPS)-induced inflammation and subsequent delayed dopaminergic neurodegeneration were accompanied by up-regulation of COMT expression and activity in microglial cells as well as in perivascular cells. Interestingly, similar perivascular up-regulation of COMT expression in inflamed renal tissue was previously noted in dTGRs. These results suggest that inflammation reactions may up-regulate COMT expression. Furthermore, this increased glial and perivascular COMT activity in the central nervous system (CNS) may decrease the bioavailability of L-DOPA and be related to the motor fluctuation noted during L-DOPA therapy in PD patients.