Development and validation of a sensitive and selective UHPLC-MS/MS method for simultaneous determination of both free and total eicosapentaeonic acid and docosahexenoic acid in human plasma

A sensitive, selective, and quantitative method for the simultaneous determination of free and total eicosapentaeonic acid (EPA) and docosahexenoic acid (DHA) has been developed and validated in human plasma using fatty acid free human serum albumin as a surrogate matrix. Clean-up for free EPA and DHA employs a liquid-liquid extraction with hexane to remove plasma interferences and provide for cleaner chromatography. The method for total EPA and DHA requires a digestion of the triglycerides followed by liquid-liquid extraction with hexane. Ultra high performance liquid chromatography (UHPLC) technology on a BEH C18 stationary phase column with 1.7 μm particle size was used for chromatographic separation, coupled to tandem mass spectrometry (UHPLC-MS/MS). The method for free EPA and DHA was validated over the concentration range of 0.05-25 μg/mL, while total EPA and DHA concentration range was 0.5-250 μg/mL. The results from assay validation show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies. To our knowledge, this work represents the first UHPLC-MS/MS based method that combines both free and total EPA and DHA with a relatively small sample volume (25 μL aliquot) and a run time of 1.5 min, facilitating automation and high throughput analysis.     

Exposure Analysis of C8- and C9-Alkylphenols,Alkylphenol Ethoxylates,and Their Metabolites in Surface Water Systems within the United States

This study was conducted to develop a statistical understanding of exposures to C8- and C9-alkylphenol ethoxylates (APEO) and their metabolites (APEM) in U.S. surface waters. Concentrations of APEO/APEM (all analytes) in freshwater have been reported by 19 investigations over the past 15 years. Only limited data are available for sediments and marine ecosystems. Based on the frequency of detection in surface waters, 67% of all analytes were below their detection limits. Although maximum reported concentrations varied with time, the average and 90th centile concentrations have remained relatively constant. Examination of frequency distributions for nonylphenol (NP) concentrations indicated that 99% of the levels in fresh surface waters are below the U.S. Environmental Protection Agency's Aquatic Life Ambient Chronic Water Quality Criteria for NP (6.6 μg/L). A conservative evaluation of aggregated NPEO/NPEM concentrations suggested that 97% of the samples contained aggregate NP equivalent concentrations that are also below 6.6 μg/L. These results suggest that on a nationwide basis, the likelihood of surface water concentrations exceeding the chronic USEPA Water Quality Criterion for NP is low.     

Bioaccumulation, oxidative stress and HSP70 expression in Cyprinus carpio L. exposed to microcystin-LR under laboratory conditions

Microcystin-LR (MC-LR) produced by cyanobacteria are potent specific hepatotoxins. So far the pathogenesis of environmental MC-LR toxicity to aquatic organisms has not been fully elucidated. In the present study the accumulation of MC-LR was investigated in various organs/tissues of Cyprinus carpio L. (C. carpio) following exposure to MC-LR for 14 d at environmentally relevant concentrations (0.1 to 10 μg L(-1)). Results showed that the presence of MC-LR enhanced toxin accumulation in all investigated organs and the highest accumulation was found in the liver of fish exposed to 5.0 μg L(-1) of MC-LR. An EPR analysis indicated ·OH intensity in liver was significantly induced at 0.1 μg L(-1) of MC-LR and then restored when the MC-LR concentration was greater than 0.1 μg L(-1). After 14-day exposure, MC-LR (1.0-10.0 μg L(-1) of MC-LR) caused a pronounced promotion of glutathione S-transferase (GST) activity and a depletion of reduced glutathione (GSH) content in fish liver, which indicated that GSH was involved in detoxification of MC-LR and the conjugation reaction of MC-LR and GSH occurred. A mild oxidative damage was evidenced by the accumulation of malondialdehyde (MDA) level at 5.0 μg L(-1) of MC-LR exposure, but which was restored when the MC-LR concentration was increased to 10.0 μg L(-1). The responses of antioxidant enzymes and the induction of HSP70 expression might contribute to MC-LR tolerance of C. carpio. However, the protein phosphatase (PP) activities were strikingly inhibited in all treated groups. Thus, the overall toxicity of environmental MC-LR on C. carpio seems to be initiated in the liver via both the ROS pathway and the PP inhibition pathway, and the latter might be more important when ambient MC-LR concentration is greater than 0.1 μg L(-1). More importantly, these results can help to support the evaluation on the potential effects of MC-LR under common environmental concentrations.     

Evaluation of blood mercury and serum selenium levels in the pregnant population of the Community of Madrid,Spain

BackgroundThe main exposure route to methylmercury (MeHg) is from eating fish and shellfish containing this compound. Since 2004, women of childbearing age in Spain have been urged not to eat some species (eg, tuna, shark, and swordfish), instead choosing low-MeHg seafood as part of a healthy diet.ObjectiveTo describe maternal total blood mercury (THg) and serum selenium (Se) in a cohort of pregnant women living in Spain as it relates to fish intake during the three trimesters and to assess whether or not Spanish women of childbearing age follow the recommendations listed in fish advisories and choose fish species with lower mercury levels.MethodsWe studied 141 female volunteers of childbearing age (16–45 years), interviewing all participants about their overall eating habits and seafood intake. Hg and Se levels were tested using cold-vapor atomic absorption spectrometry (CVAAS) and electrothermal atomic absorption spectrometry (ETAAS), respectively.ResultsAverage THg levels in pregnant women were 2.89 μg/L (standard deviation [SD], 2.75 μg/L, geometric mean [GM], 2.19 μg/L), and THg GM was positively associated with fish intake. Mean Se levels in pregnant women were 73.06 μg/L (SD, 13.38 μg/L), and Se levels were found to increase with tuna intake. In 16 (12%) pregnant women, THg was higher than the level recommended by the U.S. Environmental Protection Agency (EPA) (6.4 μg/L). A positive association was also found between THg and serum Se.ConclusionWomen of childbearing age in Spain had higher THg levels than women in other Western studies. Our study observed that 12% of women had THg levels above the safety limit set by the EPA (6.4 μg/L), and 31% had levels above the relevant benchmark level of 3.5 μg/L suggested by various researchers.     

The role of ERK-RSK signaling in the proliferation of intrahepatic biliary epithelial cells exposed to microcystin-leucine arginine

Microcystin-leucine arginine (MC-LR) is a potent specific hepatotoxin produced by cyanobacteria in diverse water systems, and it has been documented to induce liver injury and hepatocarcinogenesis. However, its toxic effects on intrahepatic biliary epithelial cells have not been invested in detail. In this study, we aimed to investigate the effects of MC-LR exposure on the intrahepatic biliary epithelial cells in the liver. MC-LR was orally administered to mice at 1 μg/L, 7.5 μg/L, 15 μg/L, or 30 μg/L for 180 consecutive days for histopathological and immunoblot analysis. We observed that MC-LR can enter intrahepatic bile duct tissue and induce hyperplasia of mice. Human primary intrahepatic biliary epithelial cells (HiBECs) were cultured with various concentrations of MC-LR for 24 h, meanwhile the cell viability and proteins level were detected. Western blotting analysis revealed that MC-LR increased RSK phosphorylation via ERK signaling. RSK participated in cell proliferation and cell cycle progression. Taken together, after chronic exposure, MC-LR-treated mice exhibited abnormal bile duct hyperplasia and thickened bile duct morphology through activating the ERK-RSK signaling. These data support the potential toxic effects of MC-LR on bile duct tissue of the liver.     

Dietary omega-6 fatty acid lowering increases bioavailability of omega-3 polyunsaturated fatty acids in human plasma lipid pools

BackgroundDietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.ObjectiveTo evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, alters unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.DesignSecondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3–L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.ResultsCompared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3–L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.ConclusionDietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFAs for 12 weeks further increases n-3 PUFA plasma concentrations and reduces AA.     

Determination of puerarin in biological samples and its application to a pharmacokinetic study by flow‐injection chemiluminescence

A simple and sensitive flow injection–chemiluminescence (FI–CL) method has been developed for the determination of puerarin, based on the fact that puerarin can greatly inhibit CL of the luminol–H₂O₂–haemoglobin system. The inhibition of CL intensity was linear to the logarithm of the concentration of puerarin in the range 0.08–10.0 μg/mL (r² = 0.9912). The limit of detection was 0.05 μg/mL (3σ) and the relative standard deviation (RSD) for 1.0 μg/mL (n = 11) of puerarin solution was 1.4%. Coupled with solid‐phase extraction (SPE) as the sample pretreatment, the determination of puerarin in biological samples and a preliminary pharmocokinetic study of puerarin in rats were performed. The recoveries for plasma and urine at three different concentrations were 89.2–110.0% and 91.4–104.8%, respectively. The pharmacokinetics of puerarin in plasma of rat coincides with the two‐compartment open model. The T_1/2α, T_1/2β, CL/F, V_Z/F, AUC_{(0 – t)}, MRT_{(0 – ∞)}, T_max and C_max were 0.77 ± 0.21 h, 7.55 ± 2.64 h, 2.43 ± 1.02 L/kg/h, 11.40 ± 3.45 L/kg, 56.67 ± 10.65 mg/h/L, 5.04 ± 2.78 h, 1.00 ± 0.35 h and 19.70 ± 4.67 μg/mL, respectively. Copyright © 2011 John Wiley & Sons, Ltd.     

Development and Validation of HPLC Method for Determination of Sodium Copper Chlorophyllin – A Food Colorant and Its Application in Pharmacokinetic Study

A simple, accurate, and precise bioanalytical method was developed and validated for the determination of pharmacokinetic parameters of sodium copper chlorophyllin, a USFDA approved food additive and colorant in rat plasma. The column used was Luna^® C₁₈ 250×4.6 mm, 100 Å, having particle size 4.5 μm, and the mobile phase used was methanol (MeOH), and 10 mM ammonium acetate buffer in the ratio of 90 : 10, the flow rate was 1 ml/min, and the injection volume of 20 μL. The retention time of sodium copper chlorophyllin was obtained at 9 min. The method was found to be linear at the range of 0.50–8.00 μg mL^?1.     

Simultaneous quantification of tracheloside and trachelogenin in rat plasma using liquid chromatography/tandem mass spectrometry

We developed and validated a quantitative method for simultaneously determining the concentrations of tracheloside and trachelogenin in rat plasma. Plasma samples were prepared by liquid-liquid extraction with ethyl acetate. Isocratic chromatographic separation was performed on a reversed-phase Diamonsil C(18) column (4.6×200 mm, 5 μm). The mobile phase consisted of methanol and 10mM aqueous ammonium formate (80:20, v/v). Analyte detection was achieved by positive electrospray ionization (ESI) tandem mass spectrometry. Calibration was performed by internal standardization with glipizide, and regression curves ranging from 0.625 to 625 ng/mL were constructed for both the analytes. The intra- and inter-day precision values were below 8%, and accuracy ranged from -5.33% to 2.53% in all quality control samples. In this study, the validated method was successfully applied to determine the pharmacokinetic profile of tracheloside and trachelogenin in rat plasma after oral and intravenous administration of trachelospermi total lignans.     

Polyunsaturated fatty acids down-regulate in vitro expression of the key intestinal cholesterol absorption protein NPC1L1: no effect of monounsaturated nor saturated fatty acids

Several transporter proteins regulate intestinal cholesterol absorption. Of these proteins, NPC1L1 is a major contributor to this process. Fatty acids (FAs) modulate cholesterol absorption by a mechanism that remains unknown. We evaluate the effect of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) on the expression of NPC1L1 and others proteins associated with cholesterol absorption (SR-BI, ABCG5, ABCG8, ABCA1, CAV-1, ANX-2) in human enterocytes in vitro. The role of SREBPs, PPARs, LXR and RXR in this process was also investigated. Caco-2/TC-7 enterocytes were incubated for 24 h with a wide range of concentrations of FA–bovine serum albumin (50–300 μM). Gene expression was analyzed by quantitative real-time PCR. The NPC1L1 protein present in enterocyte membranes was analyzed using Western blot. NPC1L1 mRNA levels were reduced 35–58% by the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) (P<.05). Linoleic acid (n-6), palmitic acid and oleic acid did not affect NPC1L1 mRNA expression. ABCA1 mRNA levels were reduced 44–70% by n-6 arachidonic acid and 43–55% by n-3 EPA (P<.05). LXR and LXR+RXR agonists decreased NPC1L1 mRNA expression by 28% and 57%, respectively (P<.05). A concentration of 200 μM of EPA and DHA decreased NPC1L1 protein expression in enterocyte membranes by 58% and 59%, respectively. We have demonstrated that the PUFAs n-3 EPA and DHA down-regulate NPC1L1 mRNA expression. In addition, PUFAs also down-regulate NPC1L1 protein expression in enterocyte membranes. LXR and RXR activation induced a similar repression effect. The lipid-lowering effect of n-3 PUFAs could be mediated in part by their action at the NPC1L1 gene level.     

THF INFLUENCE OF IRRADIANCE ON THF BIOCHEMICAL COMPOSITION OF THE PRYMNESIOPHYTE ISOCHRYSIS SP. (CLONE T-ISO)1

The effects of irradiance on the biochemical composition of the prymnesiophyte microalga, Isochrysis sp. (Parke; clone T-ISO), a popular species for mariculture, were examined. Cultures were grown under a 12:12 h light: dark (L:D) regime at five irradiances ranging from 50 to 1000 μE·m ²·s^?1 and harvested at late-logarithmic phase for analysis of biochemical composition. Gross composition varied aver the range of irradiances. The highest levels of protein were present in cells from cultures grown at 100 and 250 μE·m ³·s¹, and minimum levels of carbohydrate and lipid occurred at 50 μE·m^?2·s^?1. Because the cell dry weight was reduced at lower irradiances, different trends were evident when results were expressed as percentage of dry weights. Protein percentages were highest at Wand 100 μE·m^?2·s^?1 and carbohydrate at 100 μE·m^?2·s^?1. The composition of amino acids did not differ over the range of irradiances. Glutamate and aspartate were always present in high proportions (9.0–13.5%); histidine. methionine, tryptophan, cystine, and hydroxy-proline were minor constituents (0.0–2.6%). Glucose was the predominant sugar in all cultures, ranging from 23.0% (50 μE·m^?2·s^?1) to 45.0% (100 μE·m^?2·s^?1) of total polysaccharide. No correlation was found between the proportion of any of the sugars and irradiance. The proportions of the lipid class components and fatty acids showed little change with irradiance. The main fatty acids were 14:0, 16:0, 16:1(n-7), 18:1(n-9), 18:3(n-3). 18:4(n-3), 18:5(n-3), and 22:6(n-3). Proportions of 22: 6(n-3) increased, whereas l8:3(n-3). 18:3(n-6). and 18:4(n-3) decreased, with increasing irradiance. Pigment concentrations were highest in cultures grown at 50 μE·m^?2·s^?1, except for fucoxanthin and diadinoxanthin (100 μE·m^?2·s^?1). The concentrations of accessory pigments correlated with chlorophyll a, which decreased in concentration with increasing irradiance. On the basts of biochemical composition, an irradiance of 100 μE·m^?1·s^?1 (12:12 h L:D cycle)for the culture of Isochrysis sp. (clone T-ISO) may provide optimal nutritional value for maricultured animals, although feeding trials are now necessary to substantiate this.     

Protective effects of astaxanthin from Haematococcus pluvialis on the survival and oxidative stress of zebrafish embryos induced by microcystin-LR

Journal of Applied Phycology - This study evaluated the individual effects of microcystin-LR (MC-LR) with different concentrations (0, 10, 20, 30, 40 and 50 μg L?1) and the combined...     

Evaluating putative ecological drivers of microcystin spatiotemporal dynamics using metabarcoding and environmental data

Microcystin is a cyanobacterial hepatotoxin of global concern. Understanding the environmental factors that cause high concentrations of microcystin is crucial to the development of lake management strategies that minimize harmful exposures. While the literature is replete with studies linking cyanobacterial production of microcystin to changes in various nutrients, abiotic stressors, grazers, and competitors, no single biotic or abiotic factor has been shown to be reliably predictive of microcystin concentrations in complex ecosystems. We performed random forest regression analyses with 16S and 18S rRNA gene sequencing data and environmental data to determine which putative ecological drivers best explained spatiotemporal variation in total microcystin and several individual congeners in a eutrophic freshwater reservoir. Model performance was best for predicting concentrations of the congener MC-LR, with ca. 88% of spatiotemporal variance explained. Most of the variance was associated with changes in the relative abundance of the cyanobacterial genus Microcystis. Follow-up RF regression analyses revealed that factors that were the most important in predicting MC-LR were also the most important in predicting Microcystis population dynamics. We discuss how these results relate to prevailing ecological hypotheses regarding the function of microcystin.     

The endocrine disruption potential of betamethasone using Japanese medaka as a fish model

The presence of synthetic glucocorticosteroids (GCs) in surface water and their potential endocrine disruption (ED) activity at environmental concentrations has not been fully investigated. Synthetic GCs may interfere with endogenous receptors within the hypothalamic-pituitary-gonadal (HPG) axis and disruptions of this pathway can result in decreased reproduction and/or adverse developmental effects. Betamethasone, a synthetic GC, has been on the market in the United States since the 1980's. The Pharmaceutical Assessment and Transport Evaluation model estimated betamethasone concentrations to be <0.6 ng/L in 95% of all U.S. surface waters. Concentrations of 0.01, 0.1, and 1.0 µg betamethasone/L were used in a two-generation fish full life cycle study with Japanese medaka. Gross endpoints, secondary sexual characteristics, and vitellogenin expression were evaluated. The highest concentration at which ED outcomes are not anticipated was determined to be 0.1 µg/L. The ratio of the predicted environmental concentration to the no effect concentration for ED is less than one, indicating no risk to aquatic life from environmentally relevant concentrations of betamethasone.     

Enhancement of prodigiosin synthetase (PigC) production from recombinant Escherichia coli through optimization of induction strategy and media

PigC is a synthetase that catalyzes the condensation of 4-methoxy-2,2′-bipyrrole-5-carboxyaldehyde and 2-methyl-3-amylpyrrole to produce prodigiosin, which has a wide variety of impressive biological properties. In this study, we optimized PigC production from engineered Escherichia coli BL21(DE3). Investigation of different induction strategies revealed that autoinduction was the most appropriate method for PigC expression. As a result, PigC activity was elevated to 75.7?U/mL, nearly 2.1-fold higher than that with induction by isopropy-β-d-thiogalactoside. To achieve maximum enzyme production, the automedium components were optimized. “Single-factor experiments” showed that PigC production was greatly affected by the concentrations of glucose, yeast extract, and lactose. The Box–Behnken design for response surface methodology was then used to determine the optimal concentrations of these three components. According to a statistical approach, the optimum values of the three most influential parameters were 0.73?g/L glucose, 13.17?g/L yeast extract, and 5.86?g/L lactose. In the optimized automedium, the best PigC activity was obtained at 179.3?U/mL, which was 2.4-fold higher than using the initial medium. This study maximized PigC production as a foundation for further study and future industrial application.     

检测脂肪酸化胰岛素放射免疫分析方法的建立

目的:建立一种高灵敏度、高特异性、操作简单快捷的放射免疫分析法(RIA),用于检测比格犬血浆中的脂肪酸化胰岛素。方法:所用试剂盒主要以豚抗胰岛素抗体为主要特异性试剂,利用均相竞争抑制原理,采用平衡竞争法对比格犬血浆进行直接测定。在该系统中加入未标记的标准品或样品,则标记抗原和非标记抗原将竞争有限且定量的抗体结合位点。结果:建立了检测脂肪酸化胰岛素的RIA法并进行了确证,方法的线性范围为3.125~800μU/mL,最低检测限为3.125μU/mL,批内和批间精密度分别为4.5%~7.0%和3.9%~7.6%,冻融稳定性和稀释稳定性等良好。结论:方法学确证结果表明,本研究建立的脂肪酸化胰岛素RIA检测方法符合新生物制品临床前药代动力学研究指导原则要求,可用于脂肪酸化胰岛素在临床前药代动力学试验的检测。     

A Weight of Evidence Approach to the Aquatic Hazard Assessment of Bisphenoi A

Bisphenol A (BPA; 4,4-isopropylidene diphenol) is a chemical intermediate used primarily in the production of epoxy resins and polycarbonate products. BPA has been identified in surface waters and, hence, has been the subject of considerable research into its potential effects on aquatic organisms. Available literature on the aquatic toxicity of BPA was reviewed for quality against European Union TGD and Organization of Economic Cooperation and Development GLP principles. From this review, studies of suitable quality covering numerous ecologically relevant endpoints were identified to evaluate the survival, growth, and reproductive success of aquatic organisms exposed to BPA. Those studies yielded approximately 70 no observed effect concentrations (ranging from 16 to 3640 μg/L) and lowest observed effect concentrations (160 to 11,000 μg/L) that were considered in this weight of evidence assessment. Across all data, adverse effects on survival, growth, and reproduction occurred only at concentrations of 160 μg/L and above. Secondary biochemical (e.g., vitellogenin induction) and morphological (e.g., gonad histology) data provide insight into mechanisms of action, but do not correlate with apical endpoints related to survival, growth, and reproduction. Comparing the weight of the evidence of the aquatic toxicity data that showed chronic effects at 160 μg/L and higher with typical surface water concentrations in the range of 0.001 to 0.10 μg/ L, BPA is unlikely to cause adverse effects on aquatic populations or ecosystems.     

Determination of ticarcillin epimers in plasma and urine with high-performance liquid chromatography

A high-performance liquid chromatogaphic method was developed for determining the concentrations of ticarcillin (TIPC) epimers in human plasma and urine. Samples were prepared for HPLC analysis with a solid-phase extraction method and the concentrations of TIPC epimers were determined using reversed-phase HPLC. The mobile phase was a mixture of 0.005 M phosphate buffer (pH 7.0) and methanol (12:1, v/v) with a flow-rate of 1.0 ml/min. TIPC epimers were detected at 254 nm. Baseline separation of the two epimers was observed for both plasma and urine samples with a detection limit of ca. 1 μg/ml with a S/N ratio of 3. No peaks interfering with either of the TIPC epimers were observed on the HPLC chromatograms for blank plasma and urine. The recovery was more than 80% for both plasma and urine samples. C.V. values for intra- and inter-day variabilities were 0.9–2.1 and 1.1–6.4%, respectively, at concentrations ranging between 5 and 200 μg/ml. The present method was used to determine the concentrations of TIPC epimers in plasma and urine following intravenous injection of TIPC to a human volunteer. It was found that both epimers were actively secreted into urine and that the secretion of TIPC was not stereoselective. Plasma protein binding was also measured, which revealed stereoselective binding of TIPC in human plasma.     

Radioimmunoassay of androsterone and androsterone-3-sulfate in plasma

Details of a sensitive and specific radioimmunoassay for androsterone (1) and androsterone sulfate in plasma have been presented. Benzene extracts of plasma were chromatographed on a lumina to isolate the androsterone fraction either (a) directly after extraction (A) or (b) after solvolysis (AS). Following treatment with rabbit anti-A-17-BSA, antibody bound steroid was precipitated by ammonium sulfate. Androsterone concentrations in normal male plasma averaged 57 ± 24 (S.D.) ng/dl, range 35–135 ng/dl and for normal women, 44 ± 21 (S.D.) ng/dl, range 18–98 ng/dl. Androsterone sulfate concentrations were: males 55 ± 28 μg/dl (range 10–114 μg/dl); premenopausal females 52 ± 31 μg/dl (range 16–318 μg/dl).     

VARIABILITY OF THE HEPATOTOXIN MICROCYSTIN-LR IN HYPEREUTROPHIC DRINKING WATER LAKES1

The patterns of occurrence of the peptide hepatotoxin microcystin-LR (MC-LR) was studied in three hypereu-trophic hardwater lakes (Coal, Driedmeat, and Little Beaver) in central Alberta, Canada, over three open-water seasons. MC-LR concentration was based on high-performance liquid chromatography detection and expressed as μg.g^?1 of total plankton biomass, ng.L^?1 of lake water, and μg.g^?1 of Microcystis aeruginosa Kuetz. emend. Elenkin. MC-LR was highly variable temporally (differences up to 3 orders of magnitude) within each lake over an individual year, between years in an individual lake, and between lakes in any year. Seasonal (within-year) changes in MC-LR concentration (expressed in the preceding units) were positively correlated to the abundance and biomass Of the cyanobacterium M. aeruginosa (r =0.60–0.77), total and total dissolved phosphorus concentration (r =0.46–0.59), pH (r=0.38–0.58), and chlorophyll a (r=0.25–0.59). Surprisingly, there was no relationship between MC-LR concentration and water temperature (range: 7°-24°C, r =-0.13 to 0.02) and a negative correlation with nitrate concentration (r =–0.27 to -0.34). In two synoptic surveys examining spatial variability, MC-LR concentrations were quite variable (CV of 185 and 36% between sampling sites for Coal and Little Beaver lakes, respectively). Spatial distribution of MC-LR on any one day was correlated with the abundance and biomass of M. aeruginosa. Over a 24-h period, MC-LR concentration in M. aeruginosa decreased more than 6-fold at night relative to daytime concentrations. In general, analytical and within-site variation of MC-LR was relatively small (CV < 4 and 9%, respectively) but greatest both within and between years in a lake followed by diel and spatial variation.