The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances.

In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.

Chiral Discrimination of DL-Amino Acids by Trapped Ion Mobility Spectrometry after Derivatization with (+)-1-(9-Fluorenyl)ethyl Chloroformate.

A novel analytical method based on hybrid trapped ion mobility spectrometry-time-of-flight mass spectrometry (TIMS-TOFMS) has been developed to achieve fast enantiomeric separation of amino acids (AAs). Resolution of chiral AAs was achieved by forming diastereomers through derivatization with the chiral agent (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC), avoiding the use of reference compounds. Electrospray ionization (ESI) in positive mode yielded sodiated FLEC-AAs ions of which the diastereomers could be separated by TIMS. The effect of other alkali metal ions (such as Li and K) on the enantioselectivity was studied, but chiral discrimination was only observed for Na. TIMS conditions, including voltage ramp, ramp time, and accumulation time were optimized for each AA, and collision cross sections (CCSs) were determined for all diastereomers. The migration order of the DL enantiomers was found to be dependent on the structure of the AA. The resulting TIMS resolution (K0/ΔK0) for the FLEC-AA diastereomers on average was 115, requiring a mobility (K0) difference of about 0.009 cm2/(V s) to achieve 50%-valley separation. From the 21 AAs studied, enantiomer separation was achieved for 17 AAs with mobility differences ranging from 0.009 for lysine up to 0.061 cm2/(V s) for asparagine. Moreover, the presented methodology provided mutual separation of various AAs, allowing chiral analysis of multiple AAs simultaneously which may be challenging with previous enantioselective IMS approaches. It appeared possible to fully resolve all studied DL-AAs using three distinct TIMS methods, resulting in a total MS run time of about 3 min (1 min per method) and a total analysis time (including derivatization) of less than 15 min. The method demonstrated capable to determine enantiomeric ratios down to 2.5% with detection limits for the D enantiomers in the nanomolar range. This new TIMS-based methodology opens up possibilities for easy and fast analysis of AA enantiomers.

Bioactivity Profiling of Small-Volume Samples by Nano Liquid Chromatography Coupled to Microarray Bioassaying Using High-Resolution Fractionation.

High-throughput screening platforms for the identification of bioactive compounds in mixtures have become important tools in the drug discovery process. Miniaturization of such screening systems may overcome problems associated with small sample volumes and enhance throughput and sensitivity. Here we present a new screening platform, coined picofractionation analytics, which encompasses microarray bioassays and mass spectrometry (MS) of components from minute amounts of samples after their nano liquid chromatographic (nanoLC) separation. Herein, nanoLC was coupled to a low-volume liquid dispenser equipped with pressure-fed solenoid valves, enabling 50-nL volumes of column effluent (300 nL/min) to be discretely deposited on a glass slide. The resulting fractions were dried and subsequently bioassayed by sequential printing of nL-volumes of reagents on top of the spots. Unwanted evaporation of bioassay liquids was circumvented by employing mineral oil droplets. A fluorescence microscope was used for assay readout in kinetic mode. Bioassay data were correlated to MS data obtained using the same nanoLC conditions in order to assign bioactives. The platform provides the possibility of freely choosing a wide diversity of bioassay formats, including those requiring long incubation times. The new method was compared to a standard bioassay approach, and its applicability was demonstrated by screening plasmin inhibitors and fibrinolytic bioactives from mixtures of standards and snake venoms, revealing active peptides and coagulopathic proteases.

Liquid chromatographic nanofractionation with parallel mass spectrometric detection for the screening of plasmin inhibitors and (metallo)proteinases in snake venoms.

To better understand envenoming and to facilitate the development of new therapies for snakebite victims, rapid, sensitive, and robust methods for assessing the toxicity of individual venom proteins are required. Metalloproteinases comprise a major protein family responsible for many aspects of venom-induced haemotoxicity including coagulopathy, one of the most devastating effects of snake envenomation, and is characterized by fibrinogen depletion. Snake venoms are also known to contain anti-fibrinolytic agents with therapeutic potential, which makes them a good source of new plasmin inhibitors. The protease plasmin degrades fibrin clots, and changes in its activity can lead to life-threatening levels of fibrinolysis. Here, we present a methodology for the screening of plasmin inhibitors in snake venoms and the simultaneous assessment of general venom protease activity. Venom is first chromatographically separated followed by column effluent collection onto a 384-well plate using nanofractionation. Via a post-column split, mass spectrometry (MS) analysis of the effluent is performed in parallel. The nanofractionated venoms are exposed to a plasmin bioassay, and the resulting bioassay activity chromatograms are correlated to the MS data. To study observed proteolytic activity of venoms in more detail, venom fractions were exposed to variants of the plasmin bioassay in which the assay mixture was enriched with zinc or calcium ions, or the chelating agents EDTA or 1,10-phenanthroline were added. The plasmin activity screening system was applied to snake venoms and successfully detected compounds exhibiting antiplasmin (anti-fibrinolytic) activities in the venom of Daboia russelii, and metal-dependent proteases in the venom of Crotalus basiliscus. Graphical abstract ᅟ.

Linking cytochrome P450 enzymes from Mycobacterium tuberculosis to their cognate ferredoxin partners.

Mycobacterium tuberculosis (Mtb) codes for 20 cytochrome P450 enzymes (CYPs), considered potential drug-targets due to their essential roles in bacterial viability and host infection. Catalytic activity of mycobacterial CYPs is dependent on electron transfer from a NAD (P)H-ferredoxin-reductase (FNR) and a ferredoxin (Fd). Two FNRs (FdrA and FprA) and five ferredoxins (Fdx, FdxA, FdxC, FdxD, and Rv1786) have been found in the Mtb genome. However, as of yet, the cognate redox partnerships have not been fully established. This is confounded by the fact that heterologous redox partners are routinely used to reconstitute Mtb CYP metabolism. To this end, this study aimed to biochemically characterize and identify cognate redox partnerships for Mtb CYPs. Interestingly, all combinations of FNRs and ferredoxins were active in the reduction of oxidized cytochrome c, but steady-state kinetic assays revealed FdxD as the most efficient redox partner for FdrA, whereas Fdx coupled preferably with FprA. CYP121A1, CYP124A1, CYP125A1, and CYP142A1 metabolism with the cognate redox partners was reconstituted in vitro showing an unanticipated selectivity in the requirement for electron transfer partnership, which did not necessarily correlate with proximity in the genome. This is the first description of microbial P450 metabolism in which multiple ferredoxins are functionally linked to multiple CYPs.

An in vitro model for microbial fructoselysine degradation shows substantial interindividual differences in metabolic capacities of human fecal slurries.

Fructoselysine is formed upon heating during processing of food products, and being a key intermediate in advanced glycation end product formation considered to be potentially hazardous to human health. Human gut microbes can degrade fructoselysine to yield the short chain fatty acid butyrate. However, quantitative information on these biochemical reactions is lacking, and interindividual differences therein are not well established. Anaerobic incubations with pooled and individual human fecal slurries were optimized and applied to derive quantitative kinetic information for these biochemical reactions. Of 16 individuals tested, 11 were fructoselysine metabolizers, with Vmax, Km and kcat-values varying up to 14.6-fold, 9.5-fold, and 4.4-fold, respectively. Following fructoselysine exposure, 10 of these 11 metabolizers produced significantly increased butyrate concentrations, varying up to 8.6-fold. Bacterial taxonomic profiling of the fecal samples revealed differential abundant taxa for these reactions (e.g. families Ruminococcaceae, Christenellaceae), and Ruminococcus_1 showed the strongest correlation with fructoselysine degradation and butyrate production (ρ ≥ 0.8). This study highlights substantial interindividual differences in gut microbial degradation of fructoselysine. The presented method allows for quantification of gut microbial degradation kinetics for foodborne xenobiotics, and interindividual differences therein, which can be used to refine prediction of internal exposure.

In vitro metabolism of naphthalene and its alkylated congeners by human and rat liver microsomes via alkyl side chain or aromatic oxidation.

Mineral oils are widely applied in food production and processing and may contain polycyclic aromatic hydrocarbons (PAHs). The PAHs that may be present in mineral oils are typically alkylated, and have been barely studied. Metabolic oxidation of the aromatic ring is a key step to form DNA-reactive PAH metabolites, but may be less prominent for alkylated PAHs since alkyl substituents would facilitate side chain oxidation as an alternative. The current study investigates this hypothesis of preferential side chain oxidation at the cost of aromatic oxidation using naphthalene and a series of its alkyl substituted analogues as model compounds. The metabolism was assessed by measuring metabolite formation in rat and human liver microsomal incubations using UPLC and GC-MS/MS. The presence of an alkyl side chain markedly reduced aromatic oxidation for all alkyl-substituted naphthalenes that were converted. 1-n-Dodecyl-naphthalene was not metabolized under the experimental conditions applied. With rat liver microsomes for 1-methyl-, 2-methyl-, 1-ethyl-, and 2-ethyl- naphthalene, alkyl side chain oxidation was preferred over aromatic oxidation. With human liver microsomes this was the case for 2-methyl-, and 2-ethyl-naphthalene. It is concluded that addition of an alkyl substituent in naphthalene shifts metabolism in favor of alkyl side chain oxidation at the cost of aromatic ring oxidation. Furthermore, alkyl side chains of 6 or more carbon atoms appeared to seriously hamper and reduce overall metabolism, metabolic conversion being no longer observed with the C12 alkyl side chain. In summary, alkylation of PAHs likely reduces their chances of aromatic oxidation and bioactivation.

Antivenom Neutralization of Coagulopathic Snake Venom Toxins Assessed by Bioactivity Profiling Using Nanofractionation Analytics.

Venomous snakebite is one of the world's most lethal neglected tropical diseases. Animal-derived antivenoms are the only standardized specific therapies currently available for treating snakebite envenoming, but due to venom variation, often this treatment is not effective in counteracting all clinical symptoms caused by the multitude of injected toxins. In this study, the coagulopathic toxicities of venoms from the medically relevant snake species Bothropsasper, Calloselasmarhodostoma, Deinagkistrodonacutus, Daboiarusselii, Echiscarinatus and Echisocellatus were assessed. The venoms were separated by liquid chromatography (LC) followed by nanofractionation and parallel mass spectrometry (MS). A recently developed high-throughput coagulation assay was employed to assess both the pro- and anticoagulant activity of separated venom toxins. The neutralization capacity of antivenoms on separated venom components was assessed and the coagulopathic venom peptides and enzymes that were either neutralized or remained active in the presence of antivenom were identified by correlating bioassay results with the MS data and with off-line generated proteomics data. The results showed that most snake venoms analyzed contained both procoagulants and anticoagulants. Most anticoagulants were identified as phospholipases A2s (PLA2s) and most procoagulants correlated with snake venom metalloproteinases (SVMPs) and serine proteases (SVSPs). This information can be used to better understand antivenom neutralization and can aid in the development of next-generation antivenom treatments.

Determination of Flavonoids and Resveratrol in Wine by Turbulent-Flow Chromatography-LC-MS.

Turbulent-flow chromatography (TFC) on-line coupled to liquid chromatography mass spectrometry (LC-MS) is used to determine flavonoids and resveratrol in different types of wines. A fully automated system was developed in which 10 mL of sample (diluted wine) was passed over a TFC column, after which the retained analytes were separated by reversed-phase LC and detected by negative ion mode atmospheric-pressure chemical ionization (APCI) MS. The method proved to be fast, non-laborious, robust and sensitive. The feasibility of the method was tested on several red, white and rose wines. Quantitation of resveratrol was possible using the standard addition procedure. Red wine showed the highest amount of resveratrol (4 mg L(-1)), while rose and white wine contained concentrations which were about ten fold lower.

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite.

Snakebite is a neglected tropical disease that causes 138,000 deaths each year. Neurotoxic snake venoms contain small neurotoxins, including three-finger toxins (3FTxs), which can cause rapid paralysis in snakebite victims by blocking postsynaptic transmission via nicotinic acetylcholine receptors (nAChRs). These toxins are typically weakly immunogenic and thus are often not effectively targeted by current polyclonal antivenom therapies. We investigated whether nAChR mimics, also known as acetylcholine binding proteins (AChBPs), could effectively capture 3FTxs and therefore be developed as a novel class of snake-generic therapeutics for combatting neurotoxic envenoming. First, we identified the binding specificities of 3FTx from various medically important elapid snake venoms to nAChR using two recombinant nAChR mimics: the AChBP from Lymnaea stagnalis and a humanized neuronal α7 version (α7-AChBP). We next characterized these AChBP-bound and unbound fractions using SDS-PAGE and mass spectrometry. Interestingly, both mimics effectively captured long-chain 3FTxs from multiple snake species but largely failed to capture the highly related short-chain 3FTxs, suggesting a high level of binding specificity. We next investigated whether nAChR mimics could be used as snakebite therapeutics. We showed that while α7-AChBP alone did not protect against Naja haje (Egyptian cobra) venom lethality in vivo, it significantly prolonged survival times when coadministered with a nonprotective dose of antivenom. Thus, nAChR mimics are capable of neutralizing specific venom toxins and may be useful adjunct therapeutics for improving the safety and affordability of existing snakebite treatments by reducing therapeutic doses. Our findings justify exploring the future development of AChBPs as potential snakebite treatments.

Nanofractionation Platform with Parallel Mass Spectrometry for Identification of CYP1A2 Inhibitors in Metabolic Mixtures.

With early assessment of inhibitory properties of drug candidates and their circulating metabolites toward cytochrome P450 enzymes, drug attrition, especially later in the drug development process, can be decreased. Here we describe the development and validation of an at-line nanofractionation platform, which was applied for screening of CYP1A2 inhibitors in Phase I metabolic mixtures. With this platform, a metabolic mixture is separated by liquid chromatography (LC), followed by parallel nanofractionation on a microtiter well plate and mass spectrometry (MS) analysis. After solvent evaporation, all metabolites present in the nanofractionated mixture are assayed utilizing a fluorescence CYP1A2 inhibition bioassay performed on the plate. Next, a bioactivity chromatogram is constructed from the bioassay results. By peak shape and retention time correlation of the bioactivity peaks with the obtained MS data, CYP1A2-bioactive inhibiting metabolites can be identified. The method correctly evaluated the potency of five CYP1A2 inhibitors. Mixtures comprising potent inhibitors of CYP1A2 or in vitro-generated metabolites of ellipticine were evaluated for their inhibitory bioactivities. In both cases, good LC separation of all compounds was achieved and bioactivity data could be accurately correlated with the parallel recorded MS data. Generation and evaluation of Phase II metabolites of hydroxylated ellipticine was also pursued.

Rapid ligand fishing for identification of acetylcholinesterase-binding peptides in snake venom reveals new properties of dendrotoxins.

Acetylcholinesterase (AChE) from Electrophorus electricus (eel) was immobilized on the surface of amino-modified paramagnetic beads to serve as a model for the development, validation and application of a new affinity-based ligand-fishing assay for the discovery of bioactive peptides from complex protein mixtures such as venoms. Nano liquid chromatography-mass spectrometry (nanoLC-MS) was used for the analysis of trapped peptides. Using enzyme-functionalized beads, the ligand-fishing assay was evaluated and optimized using a peptide reference mixture composed of one acetylcholinesterase binder (fasciculin-II) and five non-binders (mambalgin-1, angiotensin-II, bradykinin, cardiotoxin and α-bungarotoxin). As proof of concept, snake venom samples spiked with fasciculin-II demonstrated assay selectivity and sensitivity, fishing the peptide binder from complex venom solutions at concentrations as low as 1.0 µg/mL. As negative controls for method validation, venoms of four different snake species, not known to harbor AChE binding peptides, were screened and no AChE binders were detected. The applicability of the ligand fishing assay was subsequently demonstrated with venom from the black mamba, Jameson's mamba and western green mamba (Dendroaspis spp.), which have previously been reported to contain the AChE binding fasciculins. Unknown peptides (i.e. not fasciculins) with affinity to AChE were recovered from all mamba venoms tested. Tryptic digestion followed by nano-LC-MS analysis of the material recovered from black mamba venom identified the peptide with highest AChE-binding affinity as dendrotoxin-I, a pre-synaptic neurotoxin previously not known to interact with AChE. Co-incubation of AChE with various dendrotoxins in vitro revealed reduced inactivation of AChE activity over time, thus demonstrating that these toxins stabilize AChE.

High-performance liquid chromatography-mass spectrometry-based acetylcholinesterase assay for the screening of inhibitors in natural extracts.

The present paper describes a High-performance liquid chromatography-mass spectrometry (LC-MS) methodology for the screening of acetylcholinesterase (AChE) inhibitors in natural extracts. AChE activity of sample components is monitored by a post-column biochemical assay that is based on the separate, sequential mixing of AChE and acetylcholine, respectively, with the HPLC eluate. AChE inhibitors are detected by measuring a decrease of product formation using electrospray MS. Ammonium bicarbonate was used as buffer in order to achieve optimum compatibility between biochemical assay and MS detection conditions. The assay is robust and stable for over 13 h and compares favourably with other AChE assays in terms of stability and sensitivity. IC(50) values of 9-aminoacridine, galanthamine, gallamine, (-)-huperzine A and thioflavin T were determined to be 0.12, 0.38, 6.4, 0.46 and 3.2 microM, respectively. The assay was used to effectively identify an AChE inhibitor present in a crude extract of Narcissus c.v. "Bridal Crown".

A microfluidic-based enzymatic assay for bioactivity screening combined with capillary liquid chromatography and mass spectrometry.

The design and implementation of a continuous-flow microfluidic assay for the screening of (complex) mixtures for bioactive compounds is described. The microfluidic chip featured two microreactors (1.6 and 2.4 microL) in which an enzyme inhibition and a substrate conversion reaction were performed, respectively. Enzyme inhibition was detected by continuously monitoring the products formed in the enzyme-substrate reaction by electrospray ionization mass spectrometry (ESI-MS). In order to enable the screening of mixtures of compounds, the chip-based assay was coupled on-line to capillary reversed-phase high-performance liquid chromatography (HPLC) with the HPLC column being operated either in isocratic or gradient elution mode. In order to improve the detection limits of the current method, sample preconcentration based on a micro on-line solid-phase extraction column was employed. The use of electrospray MS allowed the simultaneous detection of chemical (MS spectra) and biological parameters (enzyme inhibition) of ligands eluting from the HPLC column. The present system was optimized and validated using the protease cathepsin B as enzyme of choice. Inhibition of cathepsin B is detected by monitoring three product traces, obtained by cleavage of the substrate. The two microreactors provided 32 and 36 s reaction time, respectively, which resulted in sufficient assay dynamics to enable the screening of bioactive compounds. The total flow rate was 4 microL min-1, which a 25-fold decrease was compared with a macro-scale system described earlier. Detection limits of 0.17-2.6 micromol L-1 were obtained for the screening of inhibitors, which is comparable to either microtiter plate assays or continuous-flow assays described in the literature.

An efficient analytical platform for on-line microfluidic profiling of neuroactive snake venoms towards nicotinic receptor affinity.

Venomous snakes have evolved their efficient venomous arsenals mainly to immobilize prey. The highly variable toxic peptides in these venoms target a myriad of neurotoxic and haemotoxic receptors and enzymes and comprise highly interesting candidates for drug discovery. Discovery of bioactive compounds from snake venoms, however, is a challenge to achieve. We have developed and applied a methodology to rapidly assess bioactives in a snake venom proteome. Our microfluidic platform opens up efficient and rapid profiling of venomous anti-cholinergic receptor compounds. The key advantages of our methodology are: (i) nano amounts of venom needed; and (ii) a direct correlation of selected bioaffinities with accurate mass. To achieve this, we have for the first time successfully constructed a functional post nano-LC split to MS and bioaffinity profiling. In our method, comprehensive venom profiles with accurate masses and corresponding bioaffinities are obtained in one analytical run and will subsequently allow immediate purification of bioactive peptides with LC-MS, guided by accurate masses of the bioactives only. We profiled several neurotoxic Elapidae snake venoms using our methodology in combination with the acetylcholine binding protein (AChBP) as biological target protein. The latter is a homologue of nicotinic acetylcholine receptors (nAChRs), a drug target in neurodegenerative diseases and cognitive decline such as Parkinson's and Alzheimer's, and in pain related diseases. Our methodology was evaluated and validated with high-affinity α-bungarotoxin and haemotoxic/proteolytic Vipera ammodytes venom spiked with α-bungarotoxin. Thereafter, the methodology was applied to profile the venom proteomes of Dendroaspis jamesoni kaimosae, Naja annulifera and Naja nivea. Gathering comprehensive profiling data took less than 2 h per snake venom measured. The data yielded 20 AChBP ligands of which the corresponding accurate masses were used to retrieve information from literature regarding their function and targeting specificity. We found that from these 20 ligands, 11 were previously reported on, while information on the others could not be found. From these 11 peptides, five have been reported to have nAChR affinity, while the others are reported as cytotoxic, cardiotoxic or as orphan toxin. Our methodology has the potential to aid the field of profiling complex animal venoms for drug discovery.

Analytical workflow for rapid screening and purification of bioactives from venom proteomes.

Animal venoms are important sources for finding new pharmaceutical lead molecules. We used an analytical platform for initial rapid screening and identification of bioactive compounds from these venoms followed by fast and straightforward LC-MS only guided purification to obtain bioactives for further chemical and biological studies. The analytical platform consists of a nano-LC separation coupled post-column to high-resolution mass spectrometry and parallel on-line bioaffinity profiling for the acetylcholine binding protein (AChBP) in a chip based fluorescent enhancement based bioassay. AChBP is a stable structural homologue of the extracellular ligand binding domain of the α7-nicotinic acetylcholine receptor (α7-nAChR). This receptor is an extensively studied medicinal target, previously associated with epilepsy, Alzheimer's, schizophrenia and anxiety. The workflow is demonstrated with the venom of the Naja mossambica mossambica. Two medium affinity AChBP ligands were found. After subsequent LC-MS guided purification of the respective venom peptides, the purified peptides were sequenced and confirmed as Cytotoxin 1 and 2. These peptides were not reported before to have affinity for the AChBP. The purified peptides can be used for further biological studies.

Online fluorescence enhancement assay for the acetylcholine binding protein with parallel mass spectrometric identification.

The acetylcholine binding protein (AChBP) is considered an analogue for the ligand-binding domain of neuronal nicotinic acetylcholine receptors (nAChRs). Its stability and solubility in aqueous buffer allowed the development of an online bioaffinity analysis system. For this, a tracer ligand which displays enhanced fluorescence in the binding pocket of AChBP was identified from a concise series of synthetic benzylidene anabaseines. Evaluation and optimization of the bioaffinity assay was performed in a convenient microplate reader format and subsequently transferred to the online format. The high reproducibility has the prospect of estimating the affinities of ligands from an in-house drug discovery library injected in one known concentration. Furthermore, the online bioaffinity analysis system could also be applied to mixture analysis by using gradient HPLC. This led to the possibility of affinity ranking of ligands in mixtures with parallel high-resolution mass spectrometry for compound identification.

High level of alpha1-acid glycoprotein in human seminal plasma is associated with high branching and expression of Lewis(a) groups on its glycans: supporting evidence for a prostatic origin.

BACKGROUND: Changes in concentration of seminal plasma alpha(1)-acid glycoprotein (AGP) have been studied in detail before. However, the source of high levels of AGP as well as the glycosylation of seminal plasma AGP has not been elucidated yet. METHODS: The glycosylation of AGP was studied by crossed affinity immunoelectrophoresis using fucose-specific lectins and immunostaining. Glycan structure and monosaccharide analyses were performed by high pH anion exchange chromatography with pulsed amperometric detection. Fucosyltransferases were analyzed for activity and their substrate specificity was determined. RESULTS: Two types of fucosylation were detected; Lewis(x) and Lewis(a). Lewis(a) groups were only present on AGP of individuals with a high concentration and were completely absent when the AGP concentration in seminal plasma was low. Lewis(a) expression coincides with a higher degree of branching of the glycans and a relative increased alpha4-fucosyltransferase activity. The molecular weight of all seminal plasma AGP was slightly higher than of blood plasma AGP (approx. 47 vs. 41-43 kDa). CONCLUSIONS: The results indicate that AGP in seminal fluid most likely originates from the prostate and that it is either alpha3- or alpha4-fucosylated.