Pier Giorgio Pietta

Liquid chromatography/electrospray ionization mass spectrometric characterization of flavonol glycosides in tomato extracts and human plasma.

Flavonoids continue to attract wide attention as possible very useful agents for combating free radical pathologies, i.e. the pathological states associated with free radical overproduction. Commonly used methods for the analysis of plant flavonoids include high performance liquid chromatography (HPLC) and capillary electrophoresis (CE). On the other hand, the soft-ionization approach based on electrospray ionization (ESI-MS) permits highly selective analysis of complex matrices. In this work, we examined firstly the ESI-MS behaviour of representative aglycones and glycosides of flavonols, flavones and isoflavones with the aim of suggesting a possible relationship between structure and mass spectra. Using HPLC coupled to a diode array detector (DAD) for on-line UV spectra acquisition, and in parallel to ESI-MS for mass spectra (LC/DAD-ESI-MS), we have developed methodology to observe flavonols directly in tomato puree extract. In this way, it has been possible to detect intact flavonol glycosides in tomato extracts and to characterize a flavonol trisaccharide. For the first time, using LC/ESI-MS, it has been possible to detect intact flavonol glycosides in plasma of healthy volunteers and to provide further evidence on the absorption of flavonoid glycosides after consumption of common vegetables like tomatoes.

Amide protection and amide supports in solid-phase peptide synthesis

A protecting group for side-chain amide functions, and polymeric amino-supports for use in the synthesis of C-terminal amide peptides, have been developed, and their use demonstrated in the syntheses of gastrin tetrapeptide by the solution and the solid-phase procedures.

New approach for the detection of BSH and its metabolites using capillary electrophoresis and electrospray ionization mass spectrometry

Boron neutron capture therapy is a promising binary treatment for cancer. It is based on the nuclear fission that occurs when non-radioactive 10B absorbs thermal neutrons. One of the two boron compounds currently used in clinical trials for this therapy is BSH. To ensure differentiated retention in the tumour versus normal tissue prior to treatment, routine analytical methods to determine pharmacokinetics must be available. For this purpose we have developed a new, easy and time saving approach, in which the separation of boron derivatives is performed by means of capillary electrophoresis (CE). The CE method allows analyses to be performed in short times (less than 18 min), sensitively (LOD 8 pg loaded on the capillary) quantitatively (LOQ 5 microg/ml) and with a high efficiency of separation. Moreover it is simpler than HPLC and more reproducible (intra- and inter-day values were +/-1% and +/-3%, respectively), and does not require a specific column of derivatization. Mass spectrometry analysis of boron derivatives in different samples was also performed to ensure correct attribution of the CE peaks.

High-performance liquid chromatographic assay of glycosyltransferases using flavonoids as substrate

An HPLC method for the determination of glycosyltransferase activity, alternative to the radioactive assay, is proposed. The method is suitable for following the kinetics of consecutive enzymes that yield monoglucosides, diglucosides and triglucosides, as demonstrated with a pea seedling extract containing a mixture of three glucosyltransferases using flavonoids as substrate and UDP-glucose as carbohydrate donor. In this instance the HPLC determination of the three glucosides could be accomplished after separation of the aglycones by solid extraction on a Sep-Pak C18 microcolumn. After isolation of the enzyme catalysing the production of the monoglucoside of quercetin (isoquercitrin) or kaempferol (astragalin), the kinetics of the reaction were determined by HPLC, following both the increase of the product and the disappearance of the substrate. The increasing amounts of isoquercitrin and astragalin were consistent with the decrease in the amount of aglycone measured after direct injection of the reaction mixture into the HPLC system and its elution with a less polar solvent.

Application of capillary electrophoresis at low pH to oligonucleotides quality control.

A method for oligonucleotides analysis by using capillary electrophoresis at low pH in free solution is described. It may be considered an alternative to classical analytical techniques which use basic buffers and require the presence of sieving media to separate oligonucleotides as a function of their length. On the contrary, at low pH oligo nucleotides can be separated only depending on their base composition. A large set of samples consisting of 72 synthetic oligonucleotides bearing a 5-alkylamine moiety and designed for HLA genotyping were analysed. The quality of these synthetic oligos was easily assessed, and a single base difference in oligonucleotides of equal sequence was detected. The results suggest the application of this method to the emerging field of mutation detection and single nucleotide polymorfism analysis.

Purification of NAD glycohydrolase from Neurospora crassa conidia by a polyclonal immunoadsorbent

NAD glycohydrolase from Neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. The procedure was easy, non-denaturating and suitable for repetitive use of the gel. The enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.

Characteristics of bioreactors made with urease and NAD glycohydrolase reversibly bound to immobilized antibodies

The characteristics of urease and NAD glycohydrolase, immobilized on different matrices through linkage with polyclonal antibodies, were studied for their use as enzyme reactors. The stability of the derivatives was enhanced by immobilization but some leakage of activity was found, due to the equilibrium between the free and bound enzyme. The release of the enzyme to the solution depends on the affinity constant of the antibodies and the concentration of the substrate circulating in the reactor. On the other hand the main advantage of this easy method of immobilization is the low cost replacement of the inactive enzyme with fresh catalyst.

Assay of Urea by Immobilized Urease Coupled to a Differential pH-Metera

With regard to potentiometric methods, a technique based on the differential measurement of pH with urease in homogeneous solution has been already de~ c r i b e d . ~ While the assays utilizing soluble enzymes take advantage of the homogeneous media, immobilized enzymes are generally more economic because of the possibility of reusing the same catalyst several times. Thus urease was bound4 to a number of matrices in order to obtain enzyme reactors suitable for the routine assay of urea in serum or urine. This work describes a new assay based on the differential measurement of pH following the hydrolysis of urea in serum and hemolyzed blood by means of immobilized urease. The enzyme was conjugated to Ab-antiurease immobilized on nylon-coated magnetic stirrers, and the resulting bioreactors were connected to a differential pH-meter.

Immobilized Pig Brain NAD Glycohydrolase for the Preparation of NAD Analogues

Among the enzymes whose activities are affected by pyridine nucleotides, the dehydrogenases are widely studied under the biochemical and physiological point of view. Their technological importance is based on the development of methods for clinical analyses as well as on the preparation of particular biosensors. In addition to dehydrogenases, other enzymes related to NAD and NADP play an important role in reactions where the pyridine nucleotides work as substrates instead of coenzymes. Some of these reactions are connected with pathological infections, such as bacteria toxins, whereas other enzymes, named NADases or NAD glycohydrolases, catalyze reactions normally occurring in fungi, mammalian seminal plasma, mammalian organs, and other tissues, leading to the breaking of the nicotinamide N-ribosidic bond in NAD(P). The enzymes from bacteria, fungi, and mammalian seminal plasma (EC 3.2.2.5) show a pure hydrolytic activity-5 and yield nicotinamide and adenosine diphosphate ribose (ADPR) according to the following reaction:

Automatic computation of enzyme kinetics by HPLC

Enzyme activity can be easily measured by HPLC using traces of the product itself as an internal standard. Our procedure involved the development of an equation using the experimental data obtained in the kinetic assay. The entire procedure can thus be automated and a computer program is presented here for facilitating the assay and saving time. The determination of the activity of NAD kinase is reported as an example.