Synthesis, structural characterization and effect on human granulocyte intracellular cAMP levels of abscisic acid analogs.

The phytohormone abscisic acid (ABA), in addition to regulating physiological functions in plants, is also produced and released by several mammalian cell types, including human granulocytes, where it stimulates innate immune functions via an increase of the intracellular cAMP concentration ([cAMP]i). We synthesized several ABA analogs and evaluated the structure-activity relationship, by the systematical modification of selected regions of these analogs. The resulting molecules were tested for their ability to inhibit the ABA-induced increase of [cAMP]i in human granulocytes. The analogs with modified configurations at C-2' and C-3' abrogated the ABA-induced increase of the [cAMP]i and also inhibited several pro-inflammatory effects induced by exogenous ABA on granulocytes and monocytes. Accordingly, these analogs could be suitable as novel putative anti-inflammatory compounds.

Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase.

Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4(+) T lymphocytes, CD8(+) T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only L-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by L-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

A three-dimensional traction/torsion bioreactor system for tissue engineering.

PURPOSE: The aim of this study was to design, develop and validate a simple, compact bioreactor system for tissue engineering. The resulting bioreactor was designed to achieve ease-of-use and low costs for automated cell-culturing procedures onto three-dimensional scaffolds under controlled torsion/traction regimes. METHODS: Highly porous poly-caprolactone-based scaffolds were used as substrates colonized by fibroblast cells (3T3 cell line). Constructs were placed within the cylindrical culture chamber, clumped at the ends and exposed to controlled sequences of torsional stimuli (forward/back-forward sequential cycles of 100 degrees from neutral position at a rate of 600 degrees/min) through a stepper-motor; working settings were defined via PC by an easy user-interface. Cell adhesion, morphology, cytoskeletal fiber orientation and gene expression of extracellular matrix proteins (collagen type I, tenascin C, collagen type III) were evaluated after three days of torsional stimulation in the bioreactor system. RESULTS AND CONCLUSIONS: The 3D bioreactor system was validated in terms of sterility, experimental reproducibility and flexibility. Cells adhered well onto the polymeric scaffolds. Collagen type I, tenascin C and collagen type III gene expression were significantly up-regulated when cells were cultured under torsion in the bioreactor for three days. In conclusion, we have developed a simple, efficient and versatile 3D cell-culture system to engineer ligament grafts. This system can be used either as a model to investigate mechanisms of tissue development or as a graft manufacturing system for possible clinical use in the field of regenerative medicine.

Ascorbic acid pre-treated quartz stimulates TNF-alpha release in RAW 264.7 murine macrophages through ROS production and membrane lipid peroxidation.

BACKGROUND: Inhalation of crystalline silica induces a pulmonary fibrotic degeneration called silicosis caused by the inability of alveolar macrophages to dissolve the crystalline structure of phagocytosed quartz particles. Ascorbic acid is capable of partially dissolving quartz crystals, leading to an increase of soluble silica concentration and to the generation of new radical sites on the quartz surface. The reaction is specific for the crystalline forms of silica. It has been already demonstrated an increased cytotoxicity and stronger induction of pro-inflammatory cyclooxygenase-2 (COX-2) by ascorbic acid pre-treated quartz (QA) compared to untreated quartz (Q) in the murine macrophage cell line RAW 264.7. METHODS: Taking advantage of the enhanced macrophage response to QA as compared to Q particles, we investigated the first steps of cell activation and the contribution of early signals generated directly from the plasma membrane to the production of TNF-alpha, a cytokine that activates both inflammatory and fibrogenic pathways. RESULTS: Here we demonstrate that TNF-alpha mRNA synthesis and protein secretion are significantly increased in RAW 264.7 macrophages challenged with QA as compared to Q particles, and that the enhanced response is due to an increase of intracellular ROS. Plasma membrane-particle contact, in the absence of phagocytosis, is sufficient to trigger TNF-alpha production through a mechanism involving membrane lipid peroxidation and this appears to be even more detrimental to macrophage survival than particle phagocytosis itself. CONCLUSION: Taken together these data suggest that an impairment of pulmonary macrophage phagocytosis, i.e. in the case of alcoholic subjects, could potentiate lung disease in silica-exposed individuals.

A Novel Method to Synthesize Phosphocreatine and Phosphocreatine Prodrugs.

BACKGROUND: Adenosine triphosphate (ATP) is the energy currency of the body; it takes part in various and indispensable metabolic processes for the maintenance of cell homeostasis, degrading to its hydrolysis product, adenosine diphosphate (ADP). Efficient ways to restore ATP are therefore necessary in the cells. When the cell lacks energy due to ischemic conditions or high ATP demand, phosphocreatine gives its phosphate group to ADP that converts to ATP, in a reaction catalyzed by the enzyme creatine kinase. For this reason, phosphocreatine is utilized as a pharmacological treatment in human diseases that involve a failure of the cellular energy, most notably in coronary artery disease. OBJECTIVE: Commercially available phosphocreatine is currently synthesized using different methods, each of one characterized by a rather low yield of the final product, probably due to the low reactivity of the guanylating reagent. The aim of this work is to overcome the drawbacks of the synthetic methods currently employed, devising a novel synthetic route to obtain phosphocreatine and phosphocreatine prodrugs in higher yields and purity. METHOD: To obtain a higher yield of the final product and a lower number of sub-products, this method utilizes a new guanylating agent characterized by high reactivity, endowed with a protecting group t-Boc on one of the two nitrogen atoms of the guanidinic function and a protected phosphate on the other one; that compound is then conjugated with an opportune secondary amine. The obtained product is cleaved first with acidic conditions to obtain the phosphocreatine prodrug (phosphocreatine ethyl ester) and then with an enzymatic method to obtain the phosphocreatine. RESULT: Have been obtained in good yield and purity as demonstrated by HPLC and mass spectrometry analysis. CONCLUSION: This novel synthetic route permits to obtain the phosphocreatine molecule in higher yield and purity compared to the methods currently employed with a combination of chemical and enzymatic methods.

Ascorbic acid-pretreated quartz enhances cyclo-oxygenase-2 expression in RAW 264.7 murine macrophages.

Exposure to quartz particles induces a pathological process named silicosis. Alveolar macrophages initiate the disease through their activation, which is the origin of the later dysfunctions. Ascorbic acid is known to selectively dissolve the quartz surface. During the reaction, ascorbic acid progressively disappears and hydroxyl radicals are generated from the quartz surface. These observations may be relevant to mammalian quartz toxicity, as substantial amounts of ascorbic acid are present in the lung epithelium. We studied the inflammatory response of the murine macrophage cell line RAW 264.7 incubated with ascorbic acid-treated quartz, through the expression and activity of the enzyme cyclo-oxygenase-2 (COX-2). COX-2 expression and prostaglandin secretion were enhanced in cells incubated with ascorbic acid-treated quartz. In contrast, no changes were observed in cells incubated with Aerosil OX50, an amorphous form of silica. Quantification of COX-2 mRNA showed a threefold increase in cells incubated with ascorbic acid-treated quartz compared with controls. The transcription factors, NF-kappaB, pCREB and AP-1, were all implicated in the increased inflammatory response. Reactive oxygen species (H(2)O(2) and OH(*)) were involved in COX-2 expression in this experimental model. Parallel experiments performed on rat alveolar macrophages from bronchoalveolar lavage confirmed the enhanced COX-2 expression and activity in the cells incubated with ascorbic acid-treated quartz compared with untreated quartz. In conclusion, the selective interaction with, and modification of, quartz particles by ascorbic acid may be a crucial event determining the inflammatory response of macrophages, which may subsequently develop into acute inflammation, eventually leading to the chronic pulmonary disease silicosis.

High throughput HPLC-ESI-MS method for the quantitation of dexamethasone in blood plasma.

Dexamethasone is a synthetic glucocorticoid with potent anti-inflammatory properties. However, its administration causes significant side effects, specially in long-term therapy. A new approach for limiting adverse effects consists in the slow and constant deliver of this drug, using dexamethasone-21-phosphate-loaded erythrocytes (RBC) as circulating bioreactors converting the non-diffusible dexamethasone-21-phosphate into the diffusible dexamethasone. In order to evaluate the real possibility to use this new method of administration, a simple, cheap and rapid assay was set to manage a large number of samples originating from clinical studies. Due to the sample complexity and analite polarity, electrospray mass spectrometry (MS) is the most powerful technique to achieve qualitative and quantitative data. In order to overcome the complex, time-consuming and expensive LC-MS/MS methods reported in the literature in the present work a standard fluxes HPLC-ESI-MS method was set up for quantitative evaluation of dexamethasone. Thanks to the extraction ion chromatogram (XIC) feature of the software, it was possible to obtain sharp profiles for dexamethasone (DXM) and for the employed internal standard (IS) flumethasone (FM), in spite of the extremely complicated chromatogram obtained after HPLC separation of acetonitrile extracted plasma sample, thus avoiding the use of the expensive deuterated internal standard. This enabled us to obtain a linear response curve, allowing the quantification of DXM from blood samples at the picomoles level.

Photochemical internalization of a peptide nucleic acid targeting the catalytic subunit of human telomerase.

Because peptide nucleic acids (PNAs) are poorly taken up by mammalian cells, strategies need to be developed for their intracellular delivery. In the present study, we demonstrated the possibility to efficiently release a naked PNA targeting the catalytic component of human telomerase reverse transcriptase (hTERT-PNA) into the cytoplasm of DU145 prostate cancer cells through the photochemical internalization approach. After light exposure, cells treated with the hTERT-PNA and photosensitizer TPPS(2a) showed a marked inhibition of telomerase activity and a reduced cell survival, which was not observed after treatment with hTERT-PNA alone. Moreover, in a direct comparison, photochemical internalization technology proved to be more efficient to internalize the hTERT-PNA than an HIV-Tat protein-based approach.

Inhibition of the translocated c-myc in Burkitt's lymphoma by a PNA complementary to the E mu enhancer.

In Burkitt's Lymphoma there is an up-regulation of the c-myc oncogene caused by its translocation from chromosome 8 to chromosome 14, often close to the E mu enhancer of the immunoglobulin heavy chain locus (IgH). In Burkitt's Lymphoma cells, a peptide nucleic acid complementary for a specific unique E mu intronic sequence selectively blocked the expression of the c-myc oncogene under E mu control but not of other c-myc alleles. This Peptide Nucleic Acid also inhibited IgM expression in B cells. The finding that PNAs specific for a regulatory noncoding sequence can block gene expression has important conceptual and practical implications.

Comparison of novel delivery systems for antisense peptide nucleic acids.

Peptide nucleic acids (PNAs) provide a powerful tool to study the mechanism of transcription and translation, an innovative strategy to regulate target gene expression. They have been successfully used in antisense technology, for their ability to specifically bind to messenger RNA (mRNA) targets and to inhibit translation of the target genes. However, unlike most of the DNA and RNA oligonucleotides, PNAs are poorly penetrated through the cell membrane, partially due to their uncharged property. To enhance the efficiency in PNA delivery, many strategies have been explored. We here compare the efficacy of three different delivery strategies for antisense PNA: 1) conjugation to hydrophobic peptides, 2) adsorption onto polymeric microspheres and 3) encapsulation in autologous erythrocytes. To this purpose, we designed and prepared PNA sequences able to inhibit the expression of macrophage enzymes involved in inflammatory process, i.e. nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) and tested their antisense activity in a murine macrophage cellular model. Both delivery through polymeric microspheres and encapsulation into erythrocytes allowed the antisense activity of unmodified PNAs at nanomolar concentration.

Pichia pastoris production of a prolyl 4-hydroxylase derived from Chondrosia reniformis sponge: A new biotechnological tool for the recombinant production of marine collagen.

Prolyl 4-hydroxylase (P4H) is a α2ß2 tetramer catalyzing the post-translational hydroxylation of prolines in collagen. Its recombinant production is mainly pursued to realize biotechnological tools able to generate animal contaminant-free hydroxylated collagen. One promising candidate for biomedical applications is the collagen extracted from the marine sponge Chondrosia reniformis, because of its biocompatibility and because is devoid of the health risks associated with bovine and porcine collagens. Here we report on the production and selection, by enzymatic and biomolecular analyses, of a triple transformed Pichia pastoris strain expressing a stable P4H tetramer derived from C. reniformis sponge and a hydroxylated non fibrillar procollagen polypeptide from the same animal. The percentage of recombinant procollagen hydroxylated prolines inside the transformed yeast was of 36.3% analyzed by mass spectrometry indicating that the recombinant enzyme is active on its natural substrate inside the yeast cell host. Furthermore, the recombinant sponge P4H has the ability to hydroxylate its natural substrate in both X and Y positions in the Xaa-Yaa-Gly collagenous triplets. In conclusion this Pichia system seems ideal for high-level production of hydroxylated sponge- or marine-derived collagen polypeptides as well as of conotoxins or other marine proteins of high pharmacological interest needing this particular post-translational modification.

A marine biological underwater depuration system (MUDS) to process waste waters.

An underwater device, able to favour the sea auto-cleaning capacities, is herein described. This system, called MUDS (marine underwater depuration system), consists of a percolating filter and is placed at sea over an urban sewage outflow of a submarine pipeline. Due to the density difference, the water effluent flows through the percolating filter: this favours the mixing and a prompt recycling of organic matter, activating a marine trophic web. Rich microbenthic communities develop on the MUDS, both interstitially, inside the filter, and on the structure. The community mainly consists of ciliates, nematodes, harpacticoid copepods and polychaetes, all of which being organisms that increase the depuration efficiency by consumption of organic matter. This structure acts also as a deterrent for the illegal trawling activity in the area, and attracts large numbers of several finfish species, thus working as a fish aggregating device (FAD). It is possible to utilise this underwater device for medium littoral towns with strong differences in effluent discharges during the year, where the use of land-built effluent treatment plants is too expensive.

Nitric oxide production stimulated by the basic fibroblast growth factor requires the synthesis of ceramide.

Nitric oxide (NO) is an intracellular and intercellular mediator involved in the modulation of many physiologic and pathologic processes including the regulation of neoangiogenesis. We analyzed the effects of basic fibroblast growth factor (bFGF) on NO production in CHO-K1 cells and the intracellular mechanisms involved. bFGF induces NO production through activation of the endothelial NO synthase (eNOS), causing a subsequent increase in cGMP levels. In most systems, eNOS activation is a Ca(2+)-calmodulin-dependent process. In CHO-K1 cells, NO production by bFGF is Ca(2+) and MAP kinase independent, because it was not reverted by pretreatment with intracellular Ca(2+) chelators or MEK inhibitors. Translocation of the eNOS from the plasma membrane, where it is bound to caveolin 1, to the cytosol is the crucial step in the synthesis of NO. We demonstrate that the cytosolic translocation of eNOS is caused by increased synthesis of ceramide dependent by the bFGF activation of sphingomyelinase. Indeed, in the presence of the sphingomyelinase inhibitors D609 or desipramine, bFGF-dependent NO production is abrogated. To support this evidence we evaluated ceramide concentration using HPLC-electrospray ionization-mass spectrometry in controls and in bFGF-treated cells: after bFGF stimulation, a substantial increase in ceramide levels was observed. These data were further confirmed by the lack of NO production in response to fibroblast growth factor in fibroblasts derived from Niemann Pick patients who genetically lack the enzyme sphingomyelinase. In conclusion, ceramide in CHO-K1 cells is responsible for a novel Ca(2+)/calmodulin-independent mechanism for eNOS activation after fibroblast growth factor stimulation.

Sponge cell reactivity to various forms of silica.

Several sponge species incorporate a wide range of foreign material. Whether such material is actively selected by the sponge is controversial. Here we compare the available suspended matter and the sediment incorporated in the tissue of the demosponge Chondrosia reniformis. Field observations and laboratory experiments indicate that this species selects and incorporates only siliceous materials, in particular quartz particles and opal sponge spicules, avoiding carbonate particles. The reaction of ectosome cells of Chondrosia depends on the forms of silica: after settlement of crystalline quartz particles on the sponge surface, the pinacocytes contract uniformly, giving rise to a ruffled surface that remains throughout the incorporation of foreign material. In contrast, the opal spicules elicit a motile response in pinacocytes, which cover the spicules as a result. After incorporation, while the opal spicules remain unaltered within sponge tissue, the engulfed quartz particles are quickly etched, reduced in size, and released from the sponge. The etching of quartz particles by C. reniformis is produced by ascorbic acid, and is the first evidence of such activity from the animal kingdom. Ascorbic acid has been found to change the quartz surface features, which leads to an increased radical production and a consequent dissolution of quartz. This process does not occur on opal spicules.

Fiber diffraction study of spicules from marine sponges.

A synchrotron radiation fiber diffraction structural study of the axial filament of siliceous spicules from two species of marine sponges (the Demosponge Geodia cydonium and the Hexactinellid Scolymastra joubini) was carried out. The sharpness of the spots in the diffraction patterns indicated that the protein units in the filament of both samples were highly organized. A possible explanation is that the arrangement of the protein units is similar to that of the pores in highly ordered siliceous mesoporous materials. Nevertheless, the diffraction patterns are quite different for the two types of spicules. The pattern of G. cydonium is consistent with a regular 2D hexagonal lattice of protein units in the direction perpendicular to the spicule axis, with a repeating distance of 5.8 nm; the units are linked to form fibers along the axis. The pattern of S. joubini indicates the presence of two different 2D lattices in which the repeating protein units are inclined by +50 degrees and -50 degrees with respect to the elongation axis; the distance between the units increases to 8.4 nm. This 2D model is consistent with hexagonal packing of spirally oriented cylindrical protein units elongated along the filament axis.

New synthetic glutathione derivatives with increased antiviral activities.

A series of glutathione (GSH) derivatives with aliphatic chains of different lengths, coupled by peptides bound to the alpha-NH2 group of Glu, were synthesized. When added to several cell lines, the C6 (n-hexanoyl), C8 (n-octanoyl) and C12 (n-dodecanoyl) derivatives were toxic while the C2 (nethanoyl) and C4 (n-butanoyl) derivatives were not. Preliminary experiments were performed to investigate the potential antiviral activity of the C2 and C4 derivatives compared to GSH. The C4 derivative was the most potent and fully characterized. GSH-C4 is a poor substrate of GSH metabolizing enzymes; once oxidized by disulphide-bound formation, C4 is slowly reduced by GSH-reductase. GSH-C4 completely abrogated Sendai virus replication at 7.5 mM with an EC50 of 3.6 mM, compared to 7.5 mM for GSH. GSH-C4 completely inhibited herpes simplex virus (HSV-1) virus production in Vero cells at 10 mM, while the same dose of GSH caused only a 2.5 log10 reduction. Furthermore, the GSH-C4 treatment (7.5 mM) was able to markedly reduce the cytopathic effect of HSV-1 in Vero cells. Thus, GSH derivatives with increased hydrophobic properties are more effective antiviral agents against Sendai and HSV-1 viruses than GSH, suggesting their usefulness in antiviral therapy.

Molecular cloning of silicatein gene from marine sponge Petrosia ficiformis (Porifera, Demospongiae) and development of primmorphs as a model for biosilicification studies.

In some sponges peculiar proteins called silicateins catalyze silica polymerization in ordered structures, and their study is of high interest for possible biotechnological applications in the nanostructure industry. In this work we describe the isolation and the molecular characterization of silicatein from spicules of Petrosia ficiformis, a common Mediterranean sponge, and the development of a cellular model (primmorphs) suitable for in vitro studies of silicatein gene regulation. The spicule of P. ficiformis contains an axial filament composed of 2 insoluble proteins, of 30 and 23 kDa. The 23-kDa protein was characterized, and the full-length cDNA was cloned. The putative amino acid sequence has high homology with previously described silicateins from other sponge species and also is very similar to cathepsins, a cystein protease family. Finally, P. ficiformis primmorphs express the silicatein gene, suggesting that they should be a good model for biosilicification studies.

Primmorphs cryopreservation: a new method for long-time storage of sponge cells.

The possibility to cryopreserve cells allows for wide opportunities of flexible handling of cell cultures from different sponge species. Primmorphs model, a multicellular 3D aggregate formed by dissociated sponge cells, is considered one of the best approaches to establish sponge cell culture but, in spite of the available protocols for freezing sponge cells, there is no information regarding the ability of the latter to form primmorphs after thawing. In the present work, we demonstrate that, after a freezing and thawing cycle using dissociated Petrosia ficiformis cells as a model, cells viability was high but it was not possible to obtain primmorphs. The same protocol for cryopreservation was then used to directly freeze primmorphs. In this second case, after thawing, viability and the cellular proliferative level were similar to unfrozen standard primmorphs. Spiculogenesis in thawed primmorphs was evaluated by quantifying the silicatein gene expression level and by assaying the silica amount in the newly formed spicules, then compared with the correspondent values obtained in standard unfrozen primmorphs. Results indicate that the freezing cycle does not affect the spiculogenesis rate. Finally, the expression level of heat shock protein 70, a well-known stress marker, was assayed and the results showed no differences between frozen and unfrozen samples. These findings are likely to promote relevant improvements in sponge cell culture technique, allowing for a worldwide exchange of living biological material, paving the way for cell banking of Porifera.

Molecular characterization of a nonfibrillar collagen from the marine sponge Chondrosia reniformis Nardo 1847 and positive effects of soluble silicates on its expression.

We report here the complete cDNA sequence of a nonfibrillar collagen (COLch) isolated from the marine sponge Chondrosia reniformis, Nardo 1847 using a PCR approach. COLch cDNA consists of 2,563 nucleotides and includes a 5' untranslated region (UTR) of 136 nucleotides, a 3' UTR of 198 nucleotides, and an open reading frame encoding for a protein of 743 amino acids with an estimated M (r) of 72.12 kDa. The phylogenetic analysis on the deduced amino acid sequence of C-terminal end shows that the isolated sequence belongs to the short-chain spongin-like collagen subfamily, a nonfibrillar group of invertebrate collagens similar to type IV collagen. In situ hybridization analysis shows higher expression of COLch mRNA in the cortical part than in the inner part of the sponge. Therefore, COLch seems to be involved in the formation of C. reniformis ectosome, where it could play a key role in the attachment to the rocky substrata and in the selective sediment incorporation typical of these organisms. qPCR analysis of COLch mRNA level, performed on C. reniformis tissue culture models (fragmorphs), also demonstrates that this matrix protein is directly involved in sponge healing processes and that soluble silicates positively regulate its expression. These findings confirm the essential role of silicon in the fibrogenesis process also in lower invertebrates, and they should give a tool for a sustainable production of marine collagen in sponge mariculture.

Identification and structural characterization by LC-ESI-IONTRAP and LC-ESI-TOF of some metabolic conjugation products of homovanillic acid in urine of neuroblastoma patients.

The levels of urinary catecholamine metabolites, such as homovanillic acid (HVA) and vanillylmandelic acid, are routinely used as a clinical tool in the diagnosis and follow-up of neuroblastoma (NB) patients. Recently, in the Clinical Pathology Laboratory Unit of G. Gaslini Children Hospital, a commercial method that employs liquid chromatography coupled to electrochemical detection (LC-EC) has been introduced for the measurement of these metabolites in the routine laboratory practice. Using this LC-EC method, an unknown peak could be observed only in samples derived from NB patients. To investigate the nature of this peak, we used a combination of liquid chromatography-time-of-flight mass spectrometry (LC-TOF-MS) and liquid chromatography-ion trap tandem mass spectrometry (LC-IT-MS). The first approach was used to obtain the elemental composition of the ions present in this new signal. To get additional structural information useful for the elucidation of unknown compounds, the ion trap analyzer was exploited. We were able to identify not just one, but three unknown signals in urine samples from NB patients which corresponded to three conjugated products of HVA: HVA sulfate and two glucuronoconjugate isomers. The enzymatic hydrolysis with ß-glucuronidase confirmed the proposed structures, while the selective alkaline hydrolysis allowed us to distinguish the difference between phenol- and acyl-glucuronide of HVA. The latter was the unknown peak observed in LC-EC separations of urine samples from NB patients.