Marzia Galli Kienle

Uptake of S-adenosyl-L-methionine by rabbit erythrocytes.

Abstract S-[methyl- 14 C] Adenosylmethionine was incubated with rabbit blood in order to demonstrate the uptake of the whole molecule of this compound by red blood cells. The results show that the radioactivity incorporated into the cells is partially associated with S-adenosyl-L-methionine. The methyl donor activity of exogenous S-adenosyl- l -methionine is also demonstrated.

Modulation of HMG-CoA reductase activity by pantetheine/pantethine

The ability of pantetheine/pantethine to modulate the activity of HMG-CoA reductase (EC 1.1.1.34) was determined in vitro with rat liver microsomes. The decay of the activity was obtained with pantethine in the 10(-5)-10(-4) M range, whereas stimulation by pantetheine occurred at 10(-3)-10(-2) M, as previously reported for GSSG and GSH, respectively. Inhibition of HMG-CoA by pantethine in isolated liver cells was also investigated by measuring the enzyme activity in microsomes isolated from hepatocytes incubated without or with 1 mM pantethine under conditions previously shown by us to induce inhibition of cholesterol synthesis from acetate. The enzyme amount was not modified by pantethine, but in cells treated with the disulphide, the relative amounts of the thiolic active forms of the enzyme, both phosphorylated and dephosphorylated, were decreased to about half compared to controls.

Biomarkers discovery by peptide and protein profiling in biological fluids based on functionalized magnetic beads purification and mass spectrometry

Proteomics aims for the full identification and quantification of all expressed proteins in any organism. This is however an extremely tedious task since one gene often accounts for multiple proteins due to gene splicing and processing of proteins, such as the addition of post-translational modifications. Moreover, the concentration range of occurring proteins varies more than a factor of one million. For these reasons, protein profiling was considered a promising technique in the early days of proteomics. Ideally a protein profile can be observed in one single measurement. In various clinical studies profiling methods have been successful in the detection of proteome variations as a consequence of an altered homeostasis. Proteins that are differentially expressed as a consequence of a disease are very useful in medical science as they can be used as new biomarkers for the diagnosis, prognosis and as possible therapeutic targets. In order to find such proteins or biomarkers two different kinds of biological material have been used: tissue samples and body fluids. Tissues are obtained from biopsies, from stable cell lines or cell cultures, or from subcellular fractions. Despite their large usage tissues suffer from several disadvantages. Tissue samples are difficult to obtain and are comprised of several different type of cells. Standardization of the methods to obtain subcellular fraction that affects its preparation and purity is a challenge not yet solved. The difference between a cell culture and its corresponding wild type present in the body limits the translation of information derived from the first to the latter. On the contrary body fluids do not suffer from these limitations inherent to tissue samples. Fluids are very easily accessible with non- or very low-invasive methods at relatively low cost. They perfuse all the organs in the body carrying secreted protein from tissues. Therefore the protein profile of the biological fluids can reflect the status of the body. Among biological fluids serum, plasma and urine are the most analyzed samples but also cerebrospinal fluid (CSF), saliva, amniotic fluids have been used. Moreover classical methods to investigate the tissue proteome, aiming at biomarker discovery, are generally based on two-dimensional electrophoresis (2DE) and are not suitable for clinical chemistry lab requirements in which large sample cohorts have to be analyzed in a short time. This addresses another great potential of body fluids profiling: the analysis can be carried out high-throughput without sacrificing robustness and quality of the method. In fact 2DE is a laborious process that is difficult to automate. It still suffers from several technical limitations in terms of repeatability and reproducibility even though progress has been made using three different fluorescent labels that enables simultaneous migration of three samples on the same gel (e.g proteins extracted from control and disease, and the internal standard).Since the beginning of the 1990ties, when this new term (proteomics) was coined, a lot of progress has been made. Among them, several strategies to search these biomarkers in biological fluids have been developed in order to try to tackle some of the limitations of the current methods. Nowadays, mass spectrometry (MS) is the method of choice for the analysis of proteins, and as a consequence the field is now often referred to as MS-based proteomics. Direct analysis of the biological fluids with mass spectrometry is a challenging approach due to the sample complexity. To carry out a repeatable and robust mass spectrometric analysis of proteins in body fluids a suitable clean-up procedure is required in which salts and detergents are removed. The presence of salts can suppress the ionization in the mass spectrometer and chromatographic profiles may be influenced by from tailing due to co-elution of contaminants1. Therefore a pre-fractionation of the fluids is essential in order to increase the number of proteins that can be detected within a single MS-experiment, thus facilitating the discovery of new markers. Moreover, the fractionation of the biological fluids will also enrich low abundant proteins in fractions. These approaches lead to build the protein profile of the different biological fluids. Variations observed in patient profiles of body fluids compared to those of controls can be used to find the best pattern of signals that allows to discriminate two populations or to stratify the patients according to tumour stage or to the response to the therapy. One the major advantages of this strategy is that no pre-knowledge of the identity of signals selected for the cluster is needed to allow their use as biomarkers2. A specific agent to capture proteins enriches the sample and thus contributes to sensitivity enhancement. In general, protein separation techniques are based on different protein physical properties, such as size, isoelectric point, solubility and affinity. Materials known from different chromatographic platforms are coupled to the surface of a carrier in order to obtain peptides and proteins. One of the first approaches to pre-fractionate the body fluid proteome using an activated surface was the Surface-Enhanced Laser Desorption/Ionization (SELDI) technique. The SELDI technique for protein profiling is probably the most known and widely used approach in which biological fluids are applied directly to a target plate that is later introduced into a mass spectrometer. After removing unbound material to the modified surface of the SELDI chip, the molecular weight of the captured proteins on the target plate is determined using a time-of-flight (TOF) mass analyzer3. In this way the body fluid protein profile for the studied population is obtained. This technology is not free of criticism. In particular not very good reproducibility of the results due to drift, noise or the use of different lots of chips are reported. Moreover the direct identification of these markers cannot be carried out using the SELDI-TOF system. Their identity has to be determined with different analytical approaches. Promising alternatives to this technology are based on magnetic beads with a functionalized or activated surface or on miniaturized chromatographic systems that allow off-line fractionation of the proteome present in the fluids before MS analysis. The combination of magnetic bead purification and matrix-assisted laser desorption ionization (MALDI) TOF-MS has been shown a powerful alternative to the SELDI-platform: the active surface of magnetic beads is much larger, resulting in a higher binding capacity, and identification of captured peptides and protein is possible through the use of a more advanced TOF mass analyzer. Moreover, only a small part of the eluted peptide/proteins fractions are used for the protein profile and the remaining sample can be to use to identify markers with other MS-approaches (e.g. MALDI-TOF/TOF or LC-ESI-MS/MS) without the need of additional purification. This review is mainly focussed on the pre-fractionation based on magnetic beads and their applications.

Correlation between plasma levels of 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic patients.

BACKGROUND/AIM Hepatic bile acid synthesis is the main mechanism whereby the organism can degrade cholesterol. Plasma levels of 7alpha-hydroxy-4-cholesten-3-one have been reported to reflect bile acid synthesis and the expression or activity of the limiting enzyme of the main biosynthetic pathway, cholesterol 7alpha-hydroxylase. Aim of this study was to correlate the levels of this metabolite with the rates of cholesterol 7alpha-hydroxylation in vivo, a direct measurement of bile acid synthesis, in hyperlipidemic patients. DESIGN Concentrations of 7alpha-hydroxy-4-cholesten-3-one were assayed by gas-liquid chromatography: mass spectrometry in plasma samples obtained in 18 patients with primary hyperlipoproteinemia who previously underwent determination of cholesterol 7alpha-hydroxylation rates in vivo by tritium release analysis. Both determinations were performed in basal conditions and after treatment with hypolipidemic drugs (the fibric acid derivatives gemfibrozil and bezafibrate, cholestyramine alone or associated with simvastatin). RESULTS Changes in plasma 7alpha-hydroxy-4-cholesten-3-one profile closely reflected in vivo cholesterol 7alpha-hydroxylation rates during treatment with fibrates, cholestyramine and cholestyramine plus simvastatin. When plotting determinations from all studies (n=40), a very strict correlation was disclosed between plasma 7alpha-hydroxy-4-cholesten-3-one and cholesterol 7alpha-hydroxylation rates (r=0.81, P<0.001). CONCLUSIONS Plasma 7alpha-hydroxy-4-cholesten-3-one closely mirrors measurements of cholesterol 7alpha-hydroxylation rates in vivo in hyperlipidemic subjects and therefore stands as a reliable marker of global bile acid synthesis. In view of the correlation observed, these data may help to interpret changes of plasma levels of this metabolite in terms of cholesterol balance quantification.

Influence of the 21-aminosteroid U74389F on ischemia-reperfusion injury in the rat

We examined the effects of the administration of 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-piperazinyl]-pregna-1,4,9( 11)-triene-3,20-dione, monomethansulfonate (U74389F), a 21-aminosteroid and so-called lazaroid, that is characterized by an inhibitory activity against iron-dependent lipid peroxidation, on ischemia-reperfusion renal injury in a rat model. After either 60 or 90 min of ischemia, plus 2 or 24 h of reperfusion, kidneys were assayed for glutathione, adenine nucleotides and lipid peroxidation products. 60 min of ischemia produced too little oxidative stress and/or too much spontaneous recovery to allow visualization of the protective effect of the drug. 90 min of ischemia followed by reperfusion induced significant glutathione oxidation, the free oxidized glutathione to total glutathione redox ratio (%) being enhanced from 4.6 +/- 0.7% before kidney clamping to 11 +/- 1 and 8.6 +/- 1.4% at 2 and 24 h reperfusion, respectively. Treatment with the lazaroid provided significant protection against this oxidation (4.9 +/- 1.05% at 24 h reperfusion). Results of lipid peroxidation confirmed the antioxidant effect of the lazaroid. In conclusion this study provides evidence for a protective role of the tested lazaroid against ischemia-reperfusion renal injury in the rat.

Hydrolysis of cyclosporin A: identification of 1,11 seco-cyclosporin A and 4,5 seco-isocyclosporin A by FAB-MS/MS

We have previously reported that treatment of CsA with aqueous HCI gives rise to the formation of a number of water-soluble compounds. Two of these were identified from their FAB-MS/MS spectra as open-chain nona- and decapeptides. We describe here the identification of two other main compounds deriving from the same treatment. Identification was rendered possible from the comparison of their FAB-MS/MS spectra with those of methyl and acetyl derivatives. The two compounds are water-soluble, open-chain undecapeptides corresponding to 1.11 seco-CsA and of 4.5 seco-isoCsA, respectively.

Intraocular and plasma kinetics of tenoxicam in rabbits.

Non-steroidal anti-inflammatory drugs (NSAID) represent potentially useful agents in the treatment of a number of ocular pathologies, but their intraocular penetration and distribution have not yet been reported. With the aim of clarifying this point, we evaluated the concentrations of the well known NSAID, tenoxicam, in the aqueous and vitreous humors of rabbits treated i.m. with the drug (7 mg/kg). The tenoxicam kinetics in these ocular fluids followed that in plasma with the time-to-peak shifted to higher values in the vitreous (1 h) as compared to that in the aqueous and plasma (40 min). AUC was also higher in the vitreous (10.4 μg· h/ml) than in the aqueous humor (2.8 μg·h/ml).

Effects of Inhibitors of Cholesterol Biosynthesis in Isolated Rat Hepatocytes

3-Hydroxy-3-methylglutaryl CoA reductase (HMG-CoA R), the microsomal enzyme catalyzing the synthesis of mevalonate from 3-hydroxy-3-methylglutaryl CoA, is known as one of 1the key enzymes in the regulation of cholesterol biosynthesis. Many authors have demonstrated that the enzyme activity is modulated by a phosphorylation-dephosphorylation reaction.1 The equilibrium between the two forms of the enzyme has been proposed as a rapid mechanism for the regulation of cholesterol synthesis. Studies in vitro for testing the inhibitory effects of oxygenated sterols and drugs have been mainly carried out using hepatocyte cultures2,3 However in these cell cultures, the HMG-CoA R was reported to be only in its dephosphorylated active form, whereas in the 4rat liver, the enzyme is present mainly as the inactive form (80%)4.