UPLC-ESI-Q-TOF/MS Based Metabolic Profiling of Protosappanin B in Rat Plasma, Bile, Feces, Urine and Intestinal Bacteria Samples.

BACKGROUND: Caesalpinia sappan L. is a traditional medicinal plant that is used to promote blood circulation and treat stroke in China. Protosappanin B (PTB) is a unique homoisoflavone compound isolated from Sappan Lignum (the heartwood of Caesalpinia sappan L). In a previous study, the metabolic fate of PTB remained unknown. OBJECTIVE: To explore whether PTB is extensively metabolized, the metabolites of PTB in bile, plasma, urine, feces, and intestinal bacteria samples in rats were investigated. METHODS: The biosamples were investigated by ultraperformance liquid chromatography combined with time-offlight mass spectrometry (UPLC-TOF-MS/MS) with MetabolitePilot software. RESULTS: 28 metabolites were identified in the biosamples: 18 metabolites in rat bile, 8 in plasma, 20 in feces, 7 in urine and 2 in intestinal bacteria samples. Both phase I and phase II metabolites were observed. Metabolite conversion occurred via 9 proposed pathways: sulfate conjugation, glucuronide conjugation, bis-glucuronide conjugation, glucose conjugation, dehydration, oxidation, hydrolysis, methylation and hydroxymethylene loss. The metabolic pathways differed among biosamples and exhibited different distributions. Among these pathways, the most important was sulfate and glucuronide conjugation. CONCLUSION: The results showed that the small intestinal and biliary routes play an important role in the clearance and excretion of PTB. The main sites of metabolism in the PTB chemical structure were the phenolic hydroxyl and the side-chains on the eight-element ring.

An occurrence of neurotoxic shellfish poisoning by consumption of gastropods contaminated with brevetoxins.

Brevetoxins were confirmed in urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) after consumption of gastropods that were recreationally harvested from an area previously affected by a Karenia brevis bloom. Several species of gastropods (Triplofusus giganteus, Sinistrofulgur sinistrum, Cinctura hunteria, Strombus alatus, Fulguropsis spirata) and one clam (Macrocallista nimbosa) from the NSP implicated gastropod collection area (Jewfish Key, Sarasota Bay, Florida) were examined for brevetoxins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA). All gastropods and the clam were contaminated with brevetoxins. Composite B-type toxin concentrations in gastropods ranged from 1.1 to 198 µg BTX-3 equiv./g by ELISA, levels likely capable of causing NSP in consumers. Several brevetoxin metabolites previously characterized in molluscan shellfish were identified in these gastropods. Brevetoxin analog profiles by ELISA were similar in the gastropod species examined. This work documents the occurrence of NSP through consumption of a type of seafood not typically monitored in Florida to protect human health, demonstrating the need to better assess and communicate the risk of NSP to gastropod harvesters in Karenia brevis endemic areas.

Tissue distribution of amino acid- and lipid-brevetoxins after intravenous administration to C57BL/6 mice.

Brevetoxins produced during algal blooms of the dinoflagellate Karenia are metabolized by shellfish into reduction, oxidation, and conjugation products. Brevetoxin metabolites comprising amino acid- and lipid conjugates account for a large proportion of the toxicity associated with the consumption of toxic shellfish. However, the disposition of these brevetoxin metabolites has not been established. Using intravenous exposure to C57BL/6 mice, we investigated the disposition in the body of three radiolabeled brevetoxin metabolites. Amino acid-brevetoxin conjugates represented by S-desoxy-BTX-B2 (cysteine-BTX-B) and lipid-brevetoxin conjugates represented by N-palmitoyl-S-desoxy-BTX-B2 were compared to dihydro-BTX-B. Tissue concentration profiles were unique to each of the brevetoxin metabolites tested, with dihydro-BTX-B being widely distributed to all tissues, S-desoxy-BTX-B2 concentrated in kidney, and N-palmitoyl-S-desoxy-BTX-B2 having the highest concentrations in spleen, liver, and lung. Elimination patterns were also unique: dihydro-BTX-B had a greater fecal versus urinary elimination, whereas urine was a more important elimination route for S-desoxy-BTX-B2, and N-palmitoyl-S-desoxy-BTX-B2 persisted in tissues and was eliminated equally in both urine and feces. The structures particular to each brevetoxin metabolite resulting from the reduction, amino acid conjugation, or fatty acid addition of BTX-B were likely responsible for these tissue-specific distributions and unique elimination patterns. These observed differences provide further insight into the contribution each brevetoxin metabolite class has to the observed potencies.

Biomarkers of neurotoxic shellfish poisoning.

Urine specimens from patients diagnosed with neurotoxic shellfish poisoning (NSP) were examined for biomarkers of brevetoxin intoxication. Brevetoxins were concentrated from urine by using solid-phase extraction (SPE), and analyzed by enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Urine extracts were fractionated by LC, and fractions analyzed for brevetoxins by ELISA. In subsequent LC-MS/MS analyses, several brevetoxin metabolites of B-type backbone were identified, with elution profiles consistent with those of ELISA. The more abundant brevetoxin metabolites in urine were characterized structurally by LC-MS/MS. With the exception of BTX-3, brevetoxin metabolites in urine differed from those found in shellfish and in shellfish meal remnants. Proposed structures of these major urinary metabolites are methylsulfoxy BTX-3, 27-epoxy BTX-3, and reduced BTX-B5. BTX-3 was found in all specimens examined. BTX-3 concentrations in urine, as determined by LC-MS/MS, correlated well with composite toxin measurements by ELISA (r(2)=0.96). BTX-3 is a useful biomarker for confirmation of clinical diagnosis of NSP.

An electrochemiluminescence-based competitive displacement immunoassay for the type-2 brevetoxins in oyster extracts.

A new competitive electrochemiluminescence-based immunoassay for the type-2 brevetoxins in oyster extracts was developed. The assay was verified by spiking known amounts of PbTx-3 into 80% methanol extracts of Gulf Coast oysters. We also provide preliminary data demonstrating that 100% acetone extracts, aqueous homogenates, and the clinical matrixes urine and serum can also be analyzed without significant matrix interferences. The assay offers the advantages of speed ( 2 h analysis time); simplicity (only 2 additions, one incubation period, and no wash steps before analysis); low limit of quantitation (conservatively, 50 pg/mL = 1 ng/g tissue equivalents); and a stable, nonradioactive label. Due to the variety of brevetoxin metabolites present and the lack of certified reference standards for liquid chromatography-mass spectrometry confirmation, a true validation of brevetoxins in shellfish extracts is not possible at this time. However, our assay correlated well with another brevetoxin immunoassay currently in use in the United States. We believe this assay could be useful as a regulatory screening tool and could support pharmacokinetic studies in animals and clinical evaluation of neurotoxic shellfish poisoning victims.