Peripheral quantitative computed tomography-derived muscle density and peripheral magnetic resonance imaging-derived muscle adiposity: precision and associations with fragility fractures in women.

PURPOSE: To determine the degree to which muscle density and fractures are explained by inter and intramuscular fat (IMF). METHODS: Women ⋝50 years of age (Hamilton, ON, Canada) had peripheral magnetic resonance imaging and peripheral quantitative computed tomography scans at 66% of the tibial length. Muscle on computed tomography images was segmented from subcutaneous fat and bone using fixed thresholds, computing muscle density. IMF was segmented from muscle within magnetic resonance images using a region-growing algorithm, computing IMF volume. Fracture history over the last 14 years was obtained. Odds ratios for fractures were determined for muscle density, adjusting for IMF volume, total hip BMD, age and body mass index. RESULTS: Women with a history of fractures were older (N=32, age:75.6±8.3 years) than those without (N=39, age: 67.0±5.2 years) (<0.01). IMF volume explained 49.3% of variance in muscle density (p<0.001). Odds for fractures were associated with lower muscle density even after adjusting for IMF volume but were attenuated after adjusting for age. CONCLUSIONS: Muscle adiposity represents only 50% of the muscle density measurement. Properties of muscle beyond its adiposity may be related to fractures, but larger and prospective studies are needed to confirm these associations.

Reproducibility of computer-assisted joint alignment measurement in OA knee radiographs.

OBJECTIVES: (1) To investigate the reproducibility of computer-assisted measurements of knee alignment angle (KA) from digitized radiographs of osteoarthritis (OA) participants requiring total knee arthroplasty (TKA) and (2) to determine whether landmark choice affects the precision of KA measurements on radiographs. METHODS: Using a custom algorithm, femoral, central, and tibial measurement-guiding rules were interactively placed on digitized posteroanterior fixed-flexion knee radiographs by mouse control and positioned according to different anatomic landmarks. The angle subtended by lines connecting these guiding rules was measured by three readers to assess interobserver, intraobserver and experience-inexperience reproducibility. Test-retest reproducibility was evaluated with duplicate radiographs from a healthy cohort. Reproducibility was assessed using root-mean square coefficients of variation (RMSCV%). The Bland-Altman method was performed on data obtained from varying anatomic landmarks (confidence interval, CI= 95%). RESULTS: From 16 healthy and 30 TKA participants, reproducibility analyses revealed a high degree of intraobserver (n=38, RMSCV=0.56%), interobserver (n=38, RMSCV=0.72%), test-retest (n=16, RMSCV=0.87%) and experience-inexperience (n=38, RMSCV=0.73%) reproducibility with variances below 1%. Varying the orientation of tibial and femoral rules according to anatomic landmarks produced a difference that exceeded an a priori limit of agreement of -1.11 degrees to +1.67 degrees. CONCLUSION: Our custom-designed software provides a robust method for measuring KAs within digitized knee radiographs. Although test-retest analyses were only performed in a healthy cohort, we anticipate a similar degree of reproducibility in an OA sample. A standardized set of anatomic landmarks employed for KA measurement is recommended since arbitrary selection of landmarks resulted in imprecise KA measurement even with a computer-assisted technique.

Quantitative analysis of subchondral sclerosis of the tibia by bone texture parameters in knee radiographs: site-specific relationships with joint space width.

OBJECTIVES: To determine the ability of radiographic bone texture (BTX) parameters to quantify subchondral tibia sclerosis and to examine clinical relevance for assessing osteoarthritis (OA) progression. We examined the relationship between BTX parameters and each of (1) location-specific joint space width (JSW) [JSW(x)] and minimum JSW (mJSW) of the affected compartment, and (2) knee alignment (KA) angle in knee radiographs of participants undergoing total knee arthroplasty (TKA). DESIGN: Digitized fixed-flexion knee radiographs were analyzed for run-length and topological BTX parameters in a subchondral region using an algorithm. Medial JSW(x) was computed at x=0.200, 0.225, 0.250 and 0.275 according to a coordinate system defined by anatomic landmarks. mJSW was determined for medial and lateral compartment lesions. KA angles were determined from radiographs using an anatomic landmark-guided algorithm. JSW measures and the magnitude of knee malalignment were each correlated with BTX parameters. Reproducibility of BTX parameters was measured by root-mean square coefficients of variation (RMSCV%). RESULTS: Run-length BTX parameters were highly reproducible (RMSCV%<1%) while topological parameters showed poorer reproducibility (>5%). In TKA participants (17 women, 13 men; age: 66+/-9 years; body mass index (BMI): 31+/-6 kg m(-2); WOMAC: 41.5+/-16.1; Kellgren-Lawrence score mode: 4), reduced trabecular spacing (Tb.Sp) and increased free ends (FE) were correlated with decreased JSW after accounting for BMI, gender and knee malalignment. These relationships were dependent on site of JSW measurement. CONCLUSION: High reproducibility in quantifying bone sclerosis using Tb.Sp and its significant relationship with JSW demonstrated potential for assessing OA progression. Increased trabecular FE and reduced porosity observed with smaller JSW suggest collapsing subchondral bone or trabecular plate perforation in advanced knee OA.

Identification of an enzymatically formed glutathione conjugate of the cyanobacterial hepatotoxin microcystin-LR: the first step of detoxication.

Cyanobacterial toxins have adverse effects on mammals, birds and fish and are being increasingly recognised as a potent stress factor and health hazard factor in aquatic ecosystems. Microcystins, cyclic heptapeptides and a main group of the cyanotoxins are mainly retained within the producer cells during cyanobacterial bloom development. However, these toxins are released into the surrounding medium by senescence and lysis of the blooms. Any toxin present could then come into contact with a wide range of aquatic organisms including phytoplankton grazers, invertebrates, fish and aquatic plants. Recent studies showed the conversion of microcystin in animal liver to a more polar compound in correlation with a depletion of the glutathione pool of the cell. The present study shows the existence of a microcystin-LR glutathione conjugate formed enzymatically via soluble glutathione S-transferase in various aquatic organisms ranging from plants (Ceratophyllum demersum), invertebrates (Dreissena polymorpha, Daphnia magna) up to fish eggs and fish (Danio rerio). The main derived conjugate was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry yielding a mass of m/z 1302, which is equivalent to the mass assumed for a glutathione microcystin-LR conjugate. This conjugate appears to be the first step in the detoxication of a cyanobacterial toxin in aquatic organisms.

Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants.

The cyclic heptapeptide, microcystin-LR, inhibits protein phosphatases 1 (PP1) and 2A (PP2A) with Ki values below 0.1 nM. Protein phosphatase 2B is inhibited 1000-fold less potently, while six other phosphatases and eight protein kinases tested are unaffected. These results are strikingly similar to those obtained with the tumour promoter okadaic acid. We establish that okadaic acid prevents the binding of microcystin-LR to PP2A, and that protein inhibitors 1 and 2 prevent the binding of microcystin-LR to PP1. We discuss the possibility that inhibition of PP1 and PP2A accounts for the extreme toxicity of microcystin-LR, and indicate its potential value in the detection and analysis of protein kinases and phosphatases.

Immuno-crossreactivity and toxicity assessment of conjugation products of the cyanobacterial toxin, microcystin-LR.

Immunoassays are increasingly used to investigate the production, properties and fates of the cyanobacterial hepatotoxic microcystins in vitro and in vivo. Responses of an ELISA immunoassay to microcystins have been determined using the authentic toxin antigen, microcystin-LR, and conjugation products between the toxin and glutathione, cysteine-glycine and cysteine. The antibodies against microcystin-LR crossreacted with the toxin conjugation products with similar affinities (96-112%) to that of microcystin-LR, when assayed at a concentration of 1 microg l(-1). Toxicity assessment of the conjugates, in comparison to microcystin-LR, indicated a reduction according to mouse bioassay. In vitro protein phosphatase inhibition assay indicated that the conjugates possessed approximately 3-9-fold lower toxicity than microcystin-LR.

Colorimetric protein phosphatase inhibition assay of laboratory strains and natural blooms of cyanobacteria: comparisons with high-performance liquid chromatographic analysis for microcystins.

Microcystins are cyclic heptapeptide hepatotoxins commonly produced by bloom-forming genera of cyanobacteria. These toxins are potent and specific inhibitors of protein phosphatases 1 and 2A. We have optimised a rapid, simple and sensitive colorimetric protein phosphatase 1 inhibition assay, utilising the activity of protein phosphatase 1 as expressed in a recombinant strain of Escherichia coli, towards the chromogenic substrate, p-nitrophenyl phosphate. A standard curve for the inhibition of protein phosphatase 1 by microcystin-LR was constructed with an IC50 of about 38 ng ml-1 and a limit of detection of 10-20 ng ml-1. Twenty-three laboratory-grown strains and 25 natural bloom samples of cyanobacteria were analysed by high-performance liquid chromatography for microcystins and by the protein phosphatase 1 inhibition assay. Agreement for the microcystin contents of the samples detected by high-performance liquid chromatography and the protein phosphatase 1 inhibition assay showed good correlation (R2 > 0.93, P < 0.0001). The suitability of the colorimetric protein phosphatase 1 inhibition assay as a screen for cyanobacterial microcystins is discussed.

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction.

The effect of organic solvents on the high performance liquid chromatography (HPLC) analysis of cylindrospermopsin using photodiode array detection was examined since organic solvents are commonly used to extract this toxin from cyanobacteria and in the mobile phase compositions used in HPLC. Increasing concentrations of methanol resulted in an increase in the UV absorbance of purified cylindrospermopsin according to spectrometry, but to a marked decrease during HPLC analysis when the concentration of this solvent was greater than 50% methanol, or when acetonitrile concentrations exceeded 30% (v/v). Precipitation of cylindrospermopsin at these high concentrations of organic solvents was not observed. Solid phase extraction methods were developed to recover the toxin from spent extracellular growth medium after laboratory culture of Cylindrospermopsis raciborskii strain CR3 as an aid to toxin purification and from spiked environmental water samples. Using C18 and polygraphite carbon cartridges in series, 100% recoveries of cylindrospermopsin were achieved for lake waters spiked at 1 micro g l(-1).

The cyanobacterium Nodularia PCC 7804, of freshwater origin, produces [L-Har2]nodularin.

The cyanobacterium Nodularia PCC 7804, an axenic, non gas-vacuolate strain from a freshwater source, produces several metabolites with cyanobacterial hepatotoxin characteristics. The most abundant is a cyclic pentapeptide, [L-Homoarginine2]nodularin. [L-Har2]nodularin is of similar toxicity, in terms of bioassay in vivo, and the inhibition of protein phosphatase-1 in vitro to nodularin, which was present in lesser amounts in the cultures.

[D-Leu1]Microcystin-LR, from the cyanobacterium Microcystis RST 9501 and from a Microcystis bloom in the Patos Lagoon estuary, Brazil.

[D-Leu1]Microcystin-LR was identified as the most abundant microcystin from a laboratory strain of the cyanobacterium Microcystis sp. isolated from a hepatotoxic Microcystis bloom from brackish waters in the Patos Lagoon estuary, southern Brazil. Toxicity of [D-Leu1]microcystin-LR, according to bioassay and protein phosphatase inhibition assay, was similar to that of the commonly-occurring microcystin-LR, which was not detectable in the Patos Lagoon laboratory isolate. This is the first report of a microcystin containing [D-Leu1] in the cyclic heptapeptide structure of these potent cyanobacterial toxins.

Losses of the cyanobacterial toxin microcystin-LR from aqueous solution by adsorption during laboratory manipulations.

The effect of plastic and methanol on the loss of microcystin-LR from solution was analysed by HPLC with photodiode array detection (HPLC-PDA). With plastic disposable pipette tips, the loss from an aqueous microcystin-LR (MC-LR) solution was 4.2% per tip operation. Using the same pipette tip, four operations were required to completely saturate a single tip with toxin. MC-LR attached to plastic pipette tips could subsequently be eluted by methanol and detected by HPLC-PDA. At methanol concentrations below 25% (v/v), recovered concentrations of MC-LR decreased significantly. Differences in MC-LR concentration were also noted by performing 50% dilution with Milli-Q water or methanol. The results are discussed in relation to the hydrophobicity of MC-LR, analytical procedures and the avoidance of toxin losses from solution during laboratory manipulations.

Identification of anatoxin-A in benthic cyanobacteria (blue-green algae) and in associated dog poisonings at Loch Insh, Scotland.

Dog deaths occurred in 1990 and 1991 after the animals drank water containing blooms of benthic cyanobacteria along the shoreline of Loch Insh, Scotland. Signs of poisoning in the affected animals and the high neurotoxicity of bloom extracts in laboratory bioassays indicated acute poisoning due to cyanobacterial neurotoxin(s). The neurotoxic blooms consisted largely of benthic Oscillatoria species which were also observed in the stomach contents of the poisoned dogs. Stomach contents were also neurotoxic in bioassays with the same signs of poisoning as the Oscillatoria blooms. The cyanobacterial alkaloid neurotoxin anatoxin-a was identified in bloom extracts and poisoned dog stomach contents by high-performance liquid chromatography and gas chromatography-mass spectrometry. A species of benthic Oscillatoria has been isolated from the neurotoxic bloom material and shown to produce anatoxin-a in laboratory culture. These findings are the first to associate anatoxin-a toxicoses with benthic, rather than planktonic, cyanobacteria. Procedures for anatoxin-a extraction and identification from the blooms and animal material are also detailed.

Retention of Microcystis aeruginosa and microcystin by salad lettuce (Lactuca sativa) after spray irrigation with water containing cyanobacteria.

Colonies and single cells of Microcystis aeruginosa and the hepatotoxin microcystin were retained by salad lettuce after growth with spray irrigation water containing the microcystin-producing cyanobacteria. These findings are discussed in terms of crop spray irrigation with water containing cyanobacteria and potential human exposure to cyanobacterial toxins via plant foods grown in such circumstances.

Toxicity of cylindrospermopsin to the brine shrimp Artemia salina: comparisons with protein synthesis inhibitors and microcystins.

The Artemia salina bioassay was successfully applied to the analysis of the hepatotoxic cyanobacterial alkaloid and protein synthesis inhibitor, cylindrospermopsin. A dose-dependent response in mortality was observed for purified cylindrospermopsin and LC(50) values decreased with time from 8.1 to 0.71 microg/ml(-1), between 24 and 72 h, respectively. Cylindrospermopsin was slightly less potent than micro cystin-LR, with similar LC(50) values on a gravimetric basis, but was more toxic to A.salina than the protein synthesis inhibitors, cycloheximide, chloramphenicol and tetracycline. Cylindrospermopsin-containing strains of the cyanobacterium Cylindrospermopsis raciborskii were found to be toxic to A.salina and the LC(50) concentration for these strains over time was greater than the LC(50) for purified cylindrospermopsin, with the exception of C. raciborskii strain CR1.

Effects of adsorption to plastics and solvent conditions in the analysis of the cyanobacterial toxin microcystin-LR by high performance liquid chromatography.

Effects of adsorption to plastics and solvent conditions in the high performance liquid chromatographic analysis of the cyanobacterial toxin microcystin-LR were investigated. Aqueous microcystin-LR readily adsorbed to the disposable polypropylene pipette tips commonly used in laboratory manipulations. This was not affected by the pH or salinity of the solution. Furthermore, dilutions of microcystin-LR in varying concentrations of methanol and acetonitrile influenced the quantification of the microcystin-LR concentration by high performance liquid chromatography.

Direct analysis of microcystins by microbore liquid chromatography electrospray ionization ion-trap tandem mass spectrometry.

Microcystins are a group of structurally similar cyclic heptapeptide hepatotoxins and tumor promoters, produced by cyanobacteria. A microbore liquid chromatography electrospray ionization ion-trap mass spectrometry (LC-ESI-ITMS) method has been developed which is capable of separating and detecting trace amounts of microcystin variants in environmental samples. Extracted water sample was loaded onto a LC trapping column and, using a column switching technique, the compounds of interest were back-flushed onto a 1-mm LC column. Structural elucidation was achieved using ion-trap with tandem mass spectrometry in the data dependent scan mode. Collision-induced dissociation to MS3 allowed tentative identification of these cyclic peptides. Full-scan LC-ESI-MS mass spectrum was obtained when 250 pg of the authentic compound was injected onto the HPLC column, which represents the detection limit for microcystin-LR. This study demonstrated that LC-ESI-ITMS is a reliable and sensitive technique for analysing trace levels of microcystins.

Uptake, effects, and metabolism of cyanobacterial toxins in the emergent reed plant Phragmites australis (cav.) trin. ex steud.

The commonly occurring cyanobacterial toxin microcystin-LR (MC-LR) was rapidly taken up by the emergent reed plant Phragmites australis with clear distribution in the different cormus parts of the plant. Highest uptake was detected in the stem, followed by the rhizome. Enzyme extracts of the rhizome system, the stem, and the leaf revealed the presence of soluble glutathione S-transferases (sGST) measured with the model substrate 1-chloro-2,4-dinitrobenzene. A significant elevation of sGST activity in the rhizome and stem parts of P. australis was detected after a 24-h exposure to 0.5 microg/L MC-LR. Rhizome, stem, and leaf tissues were also able to conjugate several microcystin toxins. However, no conjugation, either chemical nor enzymatic, was detected using the related cyanobacterial toxin nodularin as substrate. Highest glutathione S-transferase activity for the toxin substrates was detected in the pkat/mg range in the stem of P. australis. For MC-LR, a complete metabolism from the formation of a glutathione conjugate to the degradation of a cysteine conjugate in all cormus parts of the plant is reported. The stepwise degradation of the MC-LR-glutathione conjugate to a gamma-glutamylcysteine and a cysteine conjugate was demonstrated by comparison with chemically formed reference compounds and by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This is the first evidence for the uptake and metabolism of cyanobacterial toxins by an emergent aquatic macrophyte.

Analysis of cyanobacterial toxins by physicochemical and biochemical methods.

Cyanobacteria (blue-green algae) produce a wide range of low molecular weight metabolites that include potent neurotoxins, hepatotoxins, and cytotoxins. The accumulation of such toxins in freshwaters, and in brackish and marine waters presents hazards to human and animal health by a range of exposure routes. A review is presented of developments in the detection and analysis of cyanobacterial toxins, other than bioassays, including application of physicochemical, immunoassays, and enzyme-based methods. Analytical requirements are considered with reference to recently derived guideline levels for the protection of health and to the availability, or otherwise, of purified, quantitative cyanobacterial toxin standards.

Teaching styles in the faculty-resident relationship.

As part of an effort to train family physicians to be more effective teachers in family medicine, a teaching styles workshop program was developed. The aim of the program is to help physician teachers to become more flexible in matching a teaching style to a particular teaching context. The program consists of three components: (1) a scheme for classifying teaching behaviors as belonging to one of the four basic styles identified, (2) videotaped models of each of the four basic styles, and (3) structured role playing by workshop participants in which the four styles are practiced and critiqued. The program, as used in two different sets of faculty development workshops, is described and recommendations for its use by others given.

Death of a dog attributed to the cyanobacterial (blue-green algal) hepatotoxin nodularin in South Africa.

A bull terrier died after drinking water at the margin of Zeekoevlei near Cape Town. At the time, Zeekoevlei, a hypertrophic coastal lake, contained a bloom of the cyanobacteria Nodularia spumigena and Microcystis aeruginosa. The circumstances of the incident, clinical signs of poisoning and histopathology, which mainly revealed extensive liver damage, were consistent with cyanobacterial poisoning. The cyanobacterial bloom material contained 3.47 micrograms mg-1 dry weight of the pentapeptide hepatotoxin nodularin. It is inferred that the dog died of cyanobacterial hepatotoxicosis due to the ingestion of nodularin.