Potential human health risk assessment of cylindrospermopsin accumulation and depuration in lettuce and arugula

The cyanobacterial toxin cylindrospermopsin (CYN) has become a globally important secondary metabolite due to the negative effect it has on human and animal health. As a means of evaluating the risk of human exposure to CYN, the bioaccumulation and depuration of the toxin in lettuce (Lactuca sativa L.) and arugula (Eruca sativa Mill.) were investigated, after irrigation with contaminated water. The vegetables were irrigated for 7 days with CYN (3, 5 and 10 μg/L) contaminated water (bioaccumulation phase), and subsequently, irrigated for 7 days with uncontaminated distilled water (depuration phase). In general, the bioaccumulation of CYN in both vegetables decreased with increasing exposure concentration. Bioconcentration factor (BCF) of CYN increased with the progression of the experiment at 3.0 μg/L CYN, while the reverse occurred at 5 and 10 μg/L CYN. In arugula, BCF increased at all CYN exposure concentrations throughout the study. The depuration of CYN decreased with increasing exposure concentration but was highest in the plants of both species with the highest bioaccumulation of CYN. Specifically, in plants previously irrigated with water contaminated with 3, 5 and 10 μg/L CYN, the depuration of the toxin was 60.68, 27.67 and 18.52% for lettuce, and 47, 46.21 and 27.67% for arugula, respectively. Human health risks assessment revealed that the consumption of approximately 10 to 40 g of vegetables per meal will expose children and adults to 1.00-6.00 ng CYN/kg body mass for lettuce and 2.22-7.70 ng CYN/kg body mass for arugula. The irrigation of lettuce and arugula with contaminated water containing low CYN concentrations constitutes a potential human exposure route.     

Interspecific allelopathy in cyanobacteria: Cylindrospermopsin and Cylindrospermopsis raciborskii effect on the growth and metabolism of Microcystis aeruginosa

The biological role of cyanobacteria secondary metabolites is relatively unknown although several possible hypotheses have been discussed. In the following study the effect of cylindrospermopsin (CYN) and metabolites of non-CYN producing Cylindrospermopsis raciborskii strain on growth, alkaline phosphatase (ALP) activity and microcystin-LR (MC-LR) production in Microcystis aeruginosa was evaluated. Higher concentrations of CYN (10 and 50 μg L⁻¹) induced toxicity effects demonstrated by significant growth inhibition and M. aeruginosa cell necrosis. Lower concentrations of CYN (1 and 5 μg L⁻¹) slightly decreased growth rates but significantly up-regulated ALP activity. Moreover, under all studied CYN concentrations MC-LR production strongly decreased. Spent C. raciborskii medium mimicked the CYN action by inducing strong inhibition of M. aeruginosa growth and MC-LR production and through up-regulation of ALP activity. On the other hand, spent M. aeruginosa medium did not affect C. raciborskii growth and no alterations in ALP activity were observed. Co-culturing of these two species resulted in an increase of C. raciborskii contribution at the expense of M. aeruginosa. From the results we conclude that CYN can be involved in interspecific competition in cyanobacteria and that non-CYN producing C. raciborskii strains may produce a hitherto unknown bioactive compound(s) which can mimic CYN action.     

Constitutive toxin production under various nitrogen and phosphorus regimes of three ecotypes of Cylindrospermopsis raciborskii ((Wołoszyńska) Seenayya et Subba Raju)

Cylindrospermopsis raciborskii is a global invasive cyanobacterium, with some ecotypes (i.e. strains) producing the toxin cylindrospermopsin, CYN. Multiple ecotypes can co-exist, complicating prediction of toxin concentrations based on cell concentrations. This study examined the growth response and toxin production of three Australian ecotypes of C. raciborskii, two toxic (CS-505, CS-506) and one non-toxic (CS-510), to a range of nitrogen (N) and phosphorus (P) concentrations. CYN cell quota was constant under all N:P ratios and concentration conditions, indicative of a constitutive response, yet the CYN cell quota was 6-fold higher in CS-506 compared to CS-505. The ecotypes differed in response to dissolved N depletion: there was a 4-fold difference in the number of cells heterocyst mL⁻¹ between CS-505 and CS-510, while CS-506 did not produce any heterocysts and was unable to grow in N deplete conditions. Growth rates were lower for all ecotypes as [P] increased, indicative of a species with a strategy of P storage rather than increased growth. Presumably this is an adaptation to low and fluctuating P conditions. However, the negative effect of increasing [P] on growth is surprising. In contrast, increasing [N] resulted in higher growth rates across ecotypes. This study highlights the importance of understanding differences in growth and toxin production between ecotypes in response to environmental conditions in order to more effectively predict blooms and toxin yields.     

Toxin production,retention, and extracellular release by Dinophysis acuminata during extended stationary phase and culture decline

Dinophysis spp. produce diarrhetic shellfish poisoning (DSP) toxins and pectenotoxins. The extent to which the dinoflagellate cells retain their toxicity in stationary phase, a period when cells are most toxic, and their transition into cell death is not known. Here we present results on the production, recycling, retention, and release of toxins from a monoculture of Dinophysis acuminata during these two important stages. Once stationary phase was reached, cultures were divided between light and dark treatments to identify if light influenced toxin dynamics. Light was required for long-term cell maintenance (>2 months) of D. acuminata in the absence of prey, however, in the dark, cells in stationary phase survived on reserves alone for four weeks before beginning to decline. Cells maintained relatively constant levels of intracellular OA (0.39 ± 0.03 pg/cell, 0.44 ± 0.05 pg/cell), DTX1 (0.45 ± 0.09 pg/cell, 0.64 ± 0.10 pg/cell) and PTX2 (10.4 ± 1.4 pg/cell, 11.0 ± 1.9 pg/cell) in the dark and light treatments, respectively, throughout stationary phase and into culture decline. Toxin production was only apparent during late exponential and early stationary growth when cells were actively dividing. In general, the concentration of dissolved (extracellular) toxin in the medium significantly increased upon culture aging and decline; cells did not appear to be actively or passively releasing toxin during stationary phase, but rather extracellular release was likely a result of cell death. Light availability did not have an apparent effect on toxin production, quotas, or intracellular vs. extracellular distribution. Together these results suggest that a bloom of D. acuminata would retain its cellular toxicity or potency as long as the population is viable, and that cells under conditions of low light (e.g., at the boundary or below euphotic zone) and/or minimal prey could maintain toxicity for extended periods.     

Diagnosis of caseous lymphadenitis by ELISA in naturally infected goats from Venezuela

Due to the absence of previous reports, the goal of this work was to detect caseous lymphadenitis (CLA) in goat flocks from Venezuela using an indirect immunoenzymatic assay (ELISA). Eighteen farms were randomly selected in Falcon State, North-Western Venezuela. Blood samples were taken from 259 goats, 65 of them with abscesses. Experimental inoculations were made to healthy kids with 0.5 mL inocula containing 4.7 × 10⁵ of Corynebacterium pseudotuberculosis to observe the kinetics of antibody response. Immunoenzymatic assays were carried out using exotoxin of C. pseudotuberculosis as antigen. Antibody response in experimentally inoculated animals was detected 2 weeks after infection. Of 259 field goat sera, 55.98% were positive by ELISA. Of 65 goats with abscesses, 67.69% had CLA demonstrated by bacteriological methods; from these, 72.73% showed antibodies by ELISA. Of the remaining goats negative to CLA, 47.62% had antibodies by ELISA. Sensitivity was calculated in 72.73% and specificity in 67.74%. The immunoenzymatic assay applied in this research could be useful to detect CLA in naturally infected goat flocks from Venezuela.     

Development of a highly sensitive ELISA for aldosterone in mouse urine: Validation in physiological and pathophysiological states of aldosterone excess and depletion

BackgroundClinical studies have established aldosterone as a critical physiological and pathophysiological factor in salt and water homeostasis, blood pressure control and in heart failure. Genetic and physiological studies of mice are used to model these processes. A sensitive and specific assay for aldosterone is therefore needed to monitor adrenocortical activity in murine studies of renal function and cardiovascular diseases.MethodsAntibodies against aldosterone were raised in sheep as previously described. HRP-Donkey-anti-sheep IgG enzyme tracer was produced in our laboratory using the Lightning-Link HRP technique. Aldosterone ELISA protocol was validated and optimised to achieve the best sensitivity. The assay was validated by analysing the urine of mice collected under various experimental conditions designed to stimulate or suppress aldosterone in the presence of other potentially interfering steroid hormones.ResultsCross-reactivity with the steroids most likely to interfere was minimal: corticosterone = 0.0028%, cortisol = 0.0006%, DOC = 0.0048% except for 5α-dihydro-aldosterone = 1.65%. Minimum detection limit of this ELISA was 5.2 pmole/L (1.5 pg/mL). The validity of urinary aldosterone ELISA was confirmed by the excellent correlation between results obtained before and after solvent extraction and HPLC separation step (Y = 1.092X + 0.03, R² = 0.995, n = 54). Accuracy studies, parallelism and imprecision data were determined and all found to be satisfactory. Using this assay, mean urinary aldosterone levels were (i) approximately 60-fold higher in females than males mice; (ii) increased 6-fold by dietary sodium restriction; (iii) increased 10-fold by ACTH infusion and (iv) reduced by >60% in Cyp11b1 null mice.ConclusionWe describe an ELISA for urinary aldosterone that is suitable for repeated non-invasive measurements in mice. Female aldosterone levels are higher than males. Unlike humans, most aldosterone in mouse urine is not conjugated. Increased levels were noted in response to dietary sodium restriction and ACTH treatment. The sensitivity of the assay is sufficient to detect suppressed levels in mouse models of congenital adrenal hyperplasia.     

Effect of modified MRS medium on production and purification of antimicrobial peptide ST4SA produced by Enterococcus mundtii

Highest antimicrobial activity of peptide ST4SA (51,200 AU/mL) was recorded after 14 h of growth in MRS broth with optimal production at pH 6.0 or 6.5. Growth of strain ST4SA in the presence of tryptone, yeast extract, or a combination of the two, yielded 102,400 AU/mL. An increase in production of peptide ST4SA to 102,400 AU/mL was recorded in the presence of 20.0 g/L fructose, but decreased to 25,600 AU/mL in the presence of lactose (20.0 g/L) or mannose (20.0 g/L) as sole carbon source. Lower activity (25,600 AU/mL) was recorded when 2.0 g/L K₂HPO₄ was replaced by 2.0 g/L KH₂PO₄ in MRS broth. An increase of K₂HPO₄ to 10.0 g/L and 20.0 g/L resulted in higher activity (102,400 AU/mL). Addition of glycerol to MRS broth had a negative effect on peptide ST4SA production. Production of peptide ST4SA required the presence of magnesium sulphate, manganese sulphate and 5.0 g/L sodium acetate. Exclusion of tri-ammonium citrate from the medium resulted in reduction of activity to 3,200 AU/mL. Maximum activity (102,400 AU/mL) was recorded in MRS supplemented with 1.0 ppm Vit. C, DL-6,8-thioctic acid or thiamine, respectively. Growth of Listeria ivanovii susbp. ivanovii ATCC 19119 in the presence of peptide ST4SA (12,800 AU/mL) resulted in 99% cell lysis after 18 h. Improved production of peptide ST4SA was recorded in MRS broth (Biolab) pre-treated with Amberlite XAD-1180. Precipitation with ammonium sulphate, followed by gel filtration chromatography, yielded the highest level of peptide ST4SA. This paper describes the partially deproteination of growth medium to facilitate peptide ST4SA purification.     

Effect of 5-fluoro-2′-deoxyuridine on toxin production and cell cycle regulation in marine dinoflagellate,Alexandrium tamarense

The effect of metabolic inhibitor, 5-fluoro-2′-deoxyuridine (FUdR) on toxin production and the cell cycle of marine dinoflagellate, Alexandrium tamarense, was investigated. Compared to untreated cells, FUdR at 3 μM (p < 0.05) to 300 μM (p < 0.01) inhibited the cell proliferation and toxin production in a dose-dependent manner for A. tamarense cultured in modified T₁ medium. FUdR at 203 μM resulted in cell cycle arrest at the S phase at day 4 and toxigenesis was inhibited after day 2. The toxin profiles of the FUdR-treated cultures were similar to those of the control culture. These results suggest that FUdR inhibits saxitoxin (STX) biosynthesis in the early stage of the pathway. This report is the first to demonstrate the inhibition of toxin production in A. tamarense by a nucleoside analog.     

Antibody profile in symptomatic/asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected Saudi persons

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-infected persons could be symptomatic or asymptomatic. Asymptomatic and symptomatic patients can transmit SARS-CoV-2. This study aimed to study the humoral immune response in Saudis who are Covid-19 symptomatic and asymptomatic patients. We created three types of enzyme-linked immunosorbant assays (ELISAs) to reveal IgG and IgM antibodies (Abs) against SARS-CoV-2. The developed ELISAs were designed to detect Abs against SARS-CoV-2 N, S and N + S proteins. A number of Covid-19 symptomatic (1 5 3) and asymptomatic (84) RT–PCR-confirmed patient sera were used to evaluate the ELISAs and to determine the IgG and IgM antibody profile in those patients. The sensitivity and specificity of these ELISAs were evaluated using pre-Covid-19 pandemic serum samples. The results revealed the existence of anti-SARS-CoV-2 IgG and IgM Abs in Covid-19 symptomatic and asymptomatic Saudi persons. The use of SARS-CoV-2 N and S proteins in the same ELISA greatly increased the detectability of infection. In conclusion, the Covid-19 symptomatic and asymptomatic Saudi persons demonstrated both IgG and IgM antibody profile with higher titer in symptomatic patients. The use of N + S proteins as antibody capture antigens greatly increased the ELISA sensitivity.     

Potential use of nylon scouring pad cubes attachment method for pectinase production by Aspergillus niger HFD5A-1

This study investigated the novel use of scouring pad cubes as a support matrix for immobilization of fungal cell to enhance the pectinase production. Nylon scouring pad cubes were used for immobilized Aspergillus niger HFD5A-1 cells for pectinase production in flask submerge fermentation system. The enzyme activity of immobilized cell in scouring pad cubes gave higher activity compared to free cells. Various physical parameters for culture condition were studied to evaluate its effects on pectinase production. The maximum enzyme activity obtained was 11.05 U/mL on the 6th day of cultivation after using the optimized parameters of 6 scouring pad cubes, 1 × 10⁷ spores/mL of inoculum size, agitation speed of 150 rpm and incubated at 30 °C. The use of nylon scouring pad cubes gave an increment of about 335.0% of pectinase production (11.05 U/mL) compared to free cells (2.54 U/mL). The results therefore show scouring pad cubes could be a favorable carrier to immobilize the fungal cells for higher enzyme production in submerged fermentation.     

¿Existe mayor riesgo de diabetes gestacional en pacientes con disfunción tiroidea autoinmune?

ObjectiveSome recent studies have related autoimmune thyroid dysfunction and gestational diabetes (GD). The common factor for both conditions could be the existence of pro-inflammatory homeostasis. The study objective was therefore to assess whether the presence of antithyroid antibodies is related to the occurrence of GD.Material and methodsFifty-six pregnant women with serum TSH levels ≥ 2.5 mU/mL during the first trimester were retrospectively studied. Antithyroid antibodies were measured, and an O'Sullivan test was performed. GD was diagnosed based on the criteria of the Spanish Group on Diabetes and Pregnancy.ResultsPositive antithyroid antibodies were found in 21 (37.50%) women. GD was diagnosed in 15 patients, 6 of whom (10.71%) had positive antibodies, while 9 (16.07%) had negative antibodies. Data were analyzed using exact logistic regression by LogXact-8 Cytel; no statistically significant differences were found between GD patients with positive and negative autoimmunity (OR = 1.15 [95%CI = 0.28-4.51]; P = 1.00).ConclusionsThe presence of thyroid autoimmunity in women with TSH above the recommended values at the beginning of pregnancy is not associated to development of GD. However, GD prevalence was higher in these patients as compared to the Spanish general population, suggesting the need for closer monitoring in pregnant women with TSH levels ≥ 2.5 mU/mL.     

Multipurpose Love acoustic wave immunosensor for bacteria,virus or proteins detection

A multipurpose Love acoustic wave biosensor is described in this article. As mass loading is one of the main effect involved in acoustic wave sensors, a great range of biomolecules could be detected using such sensors. In this way, the antibody/antigen binding property was used to immobilise the target species. We first compared different coupling agents to link the antibodies sensitive layer to the SiO₂ sensor surface. Results showed that GPTS monolayer, allowing covalent attachment of antibodies bioreceptors, is better suited than DTSP and protein G. It permits to obtain a dense, stable and reproducible sensitive layer of antibodies. Then, different biological species with different size and shape like proteins, bacteriophages or bacteria were detected using such sensor. Different models have been chosen to validate the effective detection of a large species range: an anti-mouse antibody has been used to simulate small molecules (< 10 nm) like proteins or toxins, bacteriophage M13 for species lower than 1 μm like virus, and Escherichia coli for bacteria which are typically longer than 1 μm. Each kind of species were successfully quickly detected from few seconds for small proteins to one hour for bacteria, with detection threshold down to 4 ng/mm² for protein and 10⁶ cfu per milliliter for bacteria.     

Comparison of ELISA and SPR biosensor technology for the detection of paralytic shellfish poisoning toxins

An enzyme labeled immunosorbent assay (ELISA) and surface plasmon resonance (SPR) biosensor assay for the detection of paralytic shellfish poisoning (PSP) toxins were developed and a comparative evaluation was performed. A polyclonal antibody (BC67) used in both assay formats was raised to saxitoxin–jeffamine–BSA in New Zealand white rabbits. Each assay format was designed as an inhibition assay. Shellfish samples (n = 54) were evaluated by each method using two simple rapid extraction procedures and compared to the AOAC high performance liquid chromatography (HPLC) and the mouse bioassay (MBA). The results of each assay format were comparable with the HPLC and MBA methods and demonstrate that an antibody with high sensitivity and broad specificity to PSP toxins can be applied to different immunological techniques. The method of choice will depend on the end-users needs. The reduced manual labor and simplicity of operation of the SPR biosensor compared to ELISA, ease of sample extraction and superior real time semi-quantitative analysis are key features that could make this technology applicable in a high-throughput monitoring unit.     

Linear and branched ß(1–3) d-glucans activate but do not prime teleost macrophages in vitro and are inactivated by dilute acid: Implications for dietary immunostimulation

ß(1–3) glucans are a diverse range of carbohydrate polymers of differing lengths and structures that make up the cell walls of yeast, fungi, algae and some plants and activate innate immune responses in plants, invertebrates and higher animals. Consequently glucans are often used as dietary immunostimulants in commercial feeds for aquacultured fish species. The present study investigates the capability of purified glucans of differing structures and configurations, including curdlan, paramylon, laminarin and purified yeast ß glucan to activate innate immunity in vitro using barramundi pronephros macrophages as a model, and compares them to Zymosan, a complex mixture derived from yeast cell walls, and lipopolysaccharide from Gram negative bacteria. All of the glucans were able to stimulate respiratory burst in barramundi macrophages at concentrations of 100 μg/mL and 1000 μg/mL, with curdlan eliciting the highest respiratory burst response at 1000 μg/mL. LPS and Zymosan were the only immunostimulants tested that could prime barramundi macrophages by incubating with low concentrations (0.1 and 1 μg/mL) for 24 h before triggering respiratory burst with PMA, suggesting teleost macrophages may not prime through the glucan receptor. As glucans are used as dietary immunostimulants, the pH of the barramundi stomach was assayed for 6 h following feeding and indicated that pH was as low as 2 for up to 6 h. Treating the glucans with dilute HCl at pH 2 completely neutralised their macrophage-activating capability. These results are important as they indicate that glucans do not prime barramundi macrophages but will activate them at high concentrations. However, it is debatable whether glucans will have any effect on macrophages if administered in the diet due to the combination of high concentration required and probable hydrolysis of the polymer structures as they pass through the acid environment of the stomach.     

Development of magnetic separation and quantum dots labeled immunoassay for the detection of mercury in biological samples

A rapid and sensitive immunoassays of mercury (Hg) in biological samples was developed using quantum dots (QDs) and magnetic beads (MBs) as fluorescent and separated probes, respectively. A monoclonal antibody (mAb) that recognizes an Hg detection antigen (BSA-DTPA-Hg) complex was produced by the injection of BALB/c mice with an Hg immunizing antigen (KLH-DTPA-Hg). Then the ascites monoclonal antibodies were purified. The Hg monoclonal antibody (Hg-mAb) is conjugated with MBs to separate Hg from biological samples, and the other antibody, which is associated with QDs, is used to detect the fluorescence. The Hg in biological samples can be quantified using the relationship between the QDs fluorescence intensity and the concentration of Hg in biological samples following magnetic separation. In this method, the detection linear range is 1–1000 ng/mL, and the minimum detection limit is 1 ng/mL. The standard addition recovery rate was 94.70–101.18%. The relative standard deviation values were 2.76–7.56%. Furthermore, the Hg concentration can be detected in less than 30 min, the significant interference of other heavy metals can be avoided, and the simultaneous testing of 96 samples can be performed. These results indicate that the method could be used for rapid monitoring Hg in the body.     

Higher circulating levels of chemokines CXCL10, CCL20 and CCL22 in patients with ischemic heart disease

Recruitment of leukocytes is one of the earliest events in the pathogenesis of ischemic heart disease (IHD) and chemokines play an important role in the migration of these cells into the inflammation sites. The aim of this study was to evaluate the CXCL10, CCL20 and CCL22 levels and the single nucleotide polymorphisms (SNPs) rs4508917, rs6749704 and rs4359426 in chemokine genes in patients with IHD to clarify any association. A total of 300 patients with IHD as having acute myocardial infarction (AMI; n = 100), stable angina (SA; n = 100) or unstable angina (UA; n = 100) and 100 healthy subjects as a control group were enrolled to study. Serum samples from all participants were tested for the CXCL10, CCL20 and CCL22 levels by using ELISA. The SNPs were determined by polymerase chain reaction–restriction length polymorphism (PCR–RFLP) method. The mean serum concentrations of CXCL10, CCL20 and CCL22 in AMI patients (395.97 ± 21.20 Pg/mL, 108.38 ± 10.31 Pg/mL and 1852.58 ± 205.77 Pg/mL), SA patients (405.48 ± 27.36 Pg/mL, 90.20 ± 7.69 Pg/mL and 2322.04 ± 231.23 Pg/mL) and UA patients (396.69 ± 22.79 Pg/mL, 141.87 ± 18.10 Pg/mL and 2754.89 ± 211.70 Pg/mL) were significantly higher than in the healthy group (179.38 ± 8.85 Pg/mL, 51.92 ± 4.62 Pg/mL and 451.82 ± 23.76 Pg/mL, respectively; P < 0.001). Similarly, the serum levels of CXCL10, CCL20 and CCL22 in total IHD patients (399.38 ± 13.77 Pg/mL, 113.49 ± 7.48 Pg/mL and 2309.84 ± 126.39 Pg/mL, respectively) were also significantly higher as compared with healthy subjects (P < 0.001). The serum levels of CCL20 and CCL22 in UA patients were significantly higher than those in SA and AMI patients, respectively (P < 0.01 and P < 0.003, respectively). The serum levels of CXCL10 and CCL20 in diabetic patients were significantly higher in comparison to non-diabetic patients (P < 0.05 and P < 0.02, respectively). The serum levels of CCL22 in dyslipidemic- and obese patients were also significantly higher in comparison with non-dyslipidemic- and non-obese patients, correspondingly (P < 0.05 and P < 0.01, respectively). There were no significant differences between men and women or between patients who treated with statin, aspirin, β-blockers or angiotensin converting enzyme (ACE) inhibitors and patients without mentioned treatment regarding the levels of chemokines. The frequency of the GG genotype at SNP rs4508917 in CXCL10 gene was higher, whereas the frequency of the AA genotype at SNP rs4359426 in CCL22 gene was lower in total patients with IHD as compared with healthy subjects (P < 0.04 and P < 0.002, respectively). These results showed that the higher levels of CXCL10, CCL20 and CCL22 were associated with IHD. The serum levels of chemokines may influence by the certain traditional risk factors of IHD and some studied SNPs, but did not influence by treatment and gender of patients.     

Simple liquid chromatography method for the quantification of irinotecan and SN38 in sheep plasma: Application to in vivo pharmacokinetics after pulmonary artery chemoembolization using drug eluting beads

A rapid and simple liquid chromatography–fluorescence detection (LC–FD) method was developed and validated for the simultaneous quantification of irinotecan (CPT11) and SN38 in sheep plasma. Camptothecin (CPT) was used as the internal standard. A single step protein precipitation with acetonitrile was used for sample preparation. The separation was achieved using a 5 μm C18 column (250 mm × 4.5 mm, 5 μm) with a mobile phase composed of 36 mM sodium dihydrogen phosphate dehydrate and 4 mM sodium 1 heptane sulfonate–acetonitrile (72:28), the pH of the mobile phase was adjusted to 3. The flow rate was 1.45 mL/min and the fluorescence detection was operated at 355/515 nm (excitation/emission wavelengths). The run time was 13 min. The method was validated with respect to selectivity, extraction recovery, linearity, intra- and inter-day precision and accuracy, limit of quantification and stability. The method has a limit of quantification of 5 ng/mL for both CPT11 and SN38. The assay was linear over concentrations ranging from 5 to 5000 ng/mL and to 240 ng/mL for CPT11 and SN38, respectively. This method was used successfully to perform plasma pharmacokinetic studies of CPT11 after pulmonary artery embolization (PACE) in a sheep model. It was also validated for CPT11 and SN38 analysis in sheep lymph and human plasma.     

Quantification of 4-aminopyridine in plasma by capillary electrophoresis with electrokinetic injection

A rapid and sensitive CE method for the determination of 4-aminopyridine in human plasma using 3,4-diaminopyridine as an internal standard was developed and validated. The analytes were extracted from 0.5-mL aliquots of human plasma by liquid–liquid extraction, using 8 mL of ethyl ether, and injected electrokinetically into capillary electrophoresis equipment. The instrumental conditions were obtained and optimized by Design of Experiments (DOE – factorial and response surface model), having as factors: separation voltage, ionic strength (buffer concentration), pH and temperature. The response variables were migration time, resolution, tailing factor and drug peak area. After obtaining mathematically predicted values for the response variables with best factors combinations, these were reproduced experimentally in good agreement with predicted values. In addition to optimal separation conditions obtained by Design of Experiments, sensitivity was improved using electrokinetic injection at 10 kV for 10 s, and a capillary with 50 cm effective length and 100 μm I.D. The final instrumental conditions were voltage at 19 kV, capillary temperature at 15 °C, wavelength at 254 nm, and phosphate buffer 100 mM, pH 2.5 as the background electrolyte. This assay was linear over a concentration range of 2.5–80 ng/mL with a lower limit of quantification of 2.5 ng/mL. The relative standard deviation for the assay precision was <7% and the accuracy was >95%. This method was successfully applied to the quantification of 4-aminopyridine (4-AP) in plasma samples from patients with spinal cord injury.     

Ichthyotoxicity of four species of gymnodinioid dinoflagellates (Kareniaceae,Dinophyta) and purified karlotoxins to larval sheepshead minnow

Two species of Kareniaceae, Karlodinium veneficum (Swan and Huon River isolates) and Karlodinium conicum, and their respective purified karlotoxins (KmTx), were investigated for ichthyotoxicity on larval sheepshead minnow. Two non-karlotoxin producing species, Karenia mikimotoi and Karlodinium ballantinum were also tested. Algal treatments included live and lysed cells (homogenized and CuSO₄ treated) with fish mortalities observed from lysed Ka. veneficum and Ka. conicum but none observed from K. mikimotoi and Ka. ballantinum. The variance in ichthyotoxicity between live and lysed cells of Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) confirm that toxin is cell bound and ichthyotoxicity increases upon lysis. Ichthyotoxic blooms of Ka. veneficum in situ in the Swan River, Western Australia and Chesapeake Bay, Maryland, USA are unrelated to algal cell density as mortality was observed with low densities. In laboratory treatments, no fish mortalities were observed upon exposure to live intact cells of all four species at algal concentrations up to 2.5 × 10⁵ cells/mL in replete nutrient growth conditions. Lysed low density (3 × 10⁴ cells/mL) Ka. veneficum (Swan and Huon River) grown under P-limited nutrients caused quicker fish mortality than those cultured in replete nutrient conditions. Pure toxin isolated from Ka. veneficum (Swan and Huon River) and Ka. conicum (Southern Ocean) were toxic to sheepshead minnow larvae, with the lethal dose lowest for Km^HuonTx 2 (508.2 ng/mL), followed by Km^SwanTx 2-1 (563.2 ng/mL), and Km_conicumTx (762.4 ng/mL).     

Simultaneous quantification of venlafaxine and O-desmethylvenlafaxine in human plasma by ultra performance liquid chromatography–tandem mass spectrometry and its application in a pharmacokinetic study

A rapid, selective and sensitive ultra performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) method was developed to simultaneously determine venlafaxine (VEN) and O-desmethylvenlafaxine (ODV) in human plasma. Sample pretreatment involved a one-step extraction with diethyl ether of 0.5 mL plasma. The separation was carried out on an ACQUITY UPLC? BEH C₁₈ column with 10 mmol/L ammonium acetate and methanol as the mobile phase at a flow rate of 0.30 mL/min. The detection was performed on a triple–quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The linear calibration curves for VEN and ODV were both obtained in the concentration range of 0.200–200 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 0.200 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were less than 13% and the accuracy (relative error, R.E.) was within ±5.3% and ±3.6% for VEN and ODV. The method herein described was superior to previous methods in sensitivity and sample throughput and successfully applied to clinical pharmacokinetic study of venlafaxine sustained-release capsule in healthy male volunteers after oral administration.