Daphnia species to test the extent to which %P can explain variation in growth and competition. Further, we measured 33P kinetics  to understand the extent to which such variables improved predictions. Genotypes showed significant variation in P content, 33P kinetics and growth rate. P content alone was a poor predictor of growth rate and competitive ability. While most genotypes exhibited the typical growth penalty under P limitation, a few varied little in growth between P diets. These observations indicate that some genotypes can maintain growth under P-limited conditions by altering P use, suggesting that decomposing P content of an individual into physiological components of P kinetics will improve stoichiometric models. More generally, attention to the interplay between nutrient content and nutrient-use is required to make inferences regarding the success of genotypes in defined conditions of nutrient supply.The framework ecological stoichiometry uses elemental composition of species to make predictions about growth and competitive ability in defined elemental supply conditions. Although intraspecific differences in stoichiometry have been observed, we have yet to understand the mechanisms generating and maintaining such variation. We used variation in phosphorus (P) content within a  Their results indicated direct effects of supply stoichiometry in determining the growth of populations varying in elemental demand. Within plants, Yu et al. )Daphnia genotypes within a species reveal substantial differences in gene expression that could underlie variation in stoichiometry [Daphnia genome . Note thiometry ; maize [hiometry ; bean [2hiometry ) althougme (e.g. ). Genotyme , tis, tisDaphme (e.g. ), or lifme (e.g. ), which me . As sucDaphnia pulicaria by testing the extent to which %P predicted growth rate and competitive ability (outcome of intraspecific competition), as well as measured the 33P kinetics  in high and low P supply. There is substantial intraspecific variation in P content , and by as much as 300% in the amount of biomass produced per mg P in the body under P-limiting conditions . Such differences in P use, without any change in P content, had predictable impacts on the quantity and quality of algae via recycling by these daphniid grazers [We addressed these issues in the freshwater crustacean nt e.g. ,11) and  and Daphia (e.g. ). This via  or low phosphorus (LP 5 µmol l−1) [−2 s−1). These P supply conditions produced algae with a C : P ratio of approximately 150 (HP) and approximately 750 (LP), respectively. Daphnia pulicaria resting eggs were isolated from the 20–24 cm sediment layer (sediment dated between ca 1967 and 1977 AD) of South Center (SC) Lake, Chisago County, Minnesota   at 20°C h et al. ) and proo N or P . Ten gen2.2.Scenedesmus was filtered from experimental chemostats onto pre-combusted (550°C for 2 h) and pre-weighed GF/C filters , and dried at 60°C for 72 h. Carbon (C) content was determined using an automated CHNOS analyzer . Total phosphorus (P) content was quantified by a modified sulfuric acid digestion method (APHA 1992) and verified using a spinach standard (NIST 1570a). We then converted C and P content by mass into molar C:P for analyses.To determine algal stoichiometry, D. pulicaria raised in contrasting P environments, we raised neonates (less than 24 h old) individually in jars in 100 ml of COMBO media containing no N or P [−1 per day of either HP or LP Scenedesmus algae for a period of 5 days. The culture media was replaced each day. P content of 10 different genotypes was measured following a 5-day period under either HP or LP conditions, with three replicates per treatment. To determine D. pulicaria P content, pre-weighed samples were combusted (550°C for 2 h) and dried at 60°C for 72 h. Total phosphorus (P) content was quantified by a modified sulfuric acid digestion method (APHA 1992) and verified using a spinach standard (NIST 1570a). Each sample was a pooled sample consisting of approximately 0.05 mg. P content was calculated as per cent of Daphnia dry mass.To determine body phosphorus content in o N or P . Individ2.3.2.3.1.33P allowed us to examine elemental use on a per-atom basis. After ingestion by an organism, we were able to measure the quantity of radioisotopes acquired by the organism. Inorganic radiotracers are primarily introduced to consumers via ingestion of autotrophic periphyton [Scenedesmus algae, fresh algae, both HP and LP, were centrifuged at 3600 rpm for 30 min. The supernatant was discarded and the precipitate was resuspended in 100 ml of no N and P COMBO medium [33P (as orthophosphate) to each 100 ml algal sample. The solutions were kept on a shaker at 20°C, 16 L : 8 D cycle and incubated for 72 h to allow the algae to fully incorporate the radioisotope. Scenedesmus algae was assumed to be uniformly radiolabelled. Following 72 h, the algal solution was centrifuged at 3600 rpm for 30 min, after which the supernatant was discarded and the precipitate was resuspended in 100 ml of no N and P COMBO medium [Radioisotope assays using riphyton . TherefoO medium . We thenO medium . Two mil2.3.2.33P radiotracer assays, following DeMott, Gulati and Siewertsen [et al. [D. pulicaria were isolated in no N and P COMBO and acclimated to experimental conditions by feeding on 1 mg C l−1 of either HP or LP Scenedesmus algae daily for 72 h. On the day of the experiment, Daphnia were pooled in jars of 5, with a minimum of 3, maximum of 5 replicates and starved for 2 h. Following starvation, the Daphnia were fed 1 mg C l−1 radiolabelled algae, either HP or LP. It is important to note that 33P is a small portion of the total P in the algae; however, it captures general patterns in P processing. Additionally, not all the same animals could be used to evaluate P kinetics. Four different rates were calculated: acquisition , assimilation (40 min of feeding and 6 h of gut clearing), net incorporation (4 h of feeding) and retention (10 min of feeding and 12 h of gut clearing). When measuring retention, we changed the media every 1, 2, 4 and 8 h in order to prevent recycling. We defined acquisition as the intake of P in a given period (10 min), before having been absorbed through the gut wall. Assimilation was defined as absorption of those elements through the gut wall into body tissue, net incorporation as the amount of P allocated following assimilation, and retention as the amount of P retained after 12 h of not feeding. Daphnia thus collected were placed in scintillation vials for radioactive counting (Beckman Coulter LS 6500). The amount of radioactive 33P was calculated in each organism, correcting for mass in micrograms per microgram dry weight from an average of five separate individuals raised in identical conditions. All samples for each genotype were taken and analysed on the same day of the experiment.We performed ewertsen  and Roy  [et al. . Similar2.4.D. pulicaria individuals were fed 3 mg C l−1 per day (to elucidate greater differences in growth) of either HP or LP Scenedesmus algae, with four replicates per treatment. The culture media was replaced daily. Growth rates of 10 different genotypes were measured over a 5-day period under either HP or LP conditions. Four neonates of each genotype (less than 24 h old) were used for each P-supply treatment, separated individually in 100 ml of COMBO media containing no N or P [To determine juvenile growth rate under contrasting P supply conditions, o N or P . The len2.5.ca 2002 and 2008 AD) from SC Lake, and were used to compare competitive abilities of the experimental genotypes from the 20–24 cm layer of SC. Each reference genotype was paired with each experimental genotype for a total of 30 trials per P treatment. Neonates were placed in 500 ml flow-through polyvinyl chloride microcosms containing no N and P COMBO medium [et al. [Scenedesmus obliquus algae were pumped into microcosms one hour before adding neonates, so as to have food available upon immediate transfer. Each 500 ml microcosm vessel received 1 mg C l−1 per day of either HP or LP algae continuously for a duration of 21 days at a rate of approximately 1.725 l day−1. There was no early mortality within the first 5 days. Following the 21 days, microcosm chambers were homogenized and 30% (150 ml) of the media was sampled. Twelve Daphnia were chosen at random from a 50 ml subset of the 150 ml sample to test for unique genotype-specific  markers. Allozyme electrophoresis followed the protocols of Hebert & Beaton [Daphnia genotype in competition. Each genotype possessed a unique allozyme (i.e. electromorph) marker at the glucose-6-phosphate isomerase  5.3.1.9) locus and/or PGM (EC 5.4.2.2) locus. Two experimental genotypes (from 20–24 cm sediment layer) share the same GPI electromorph profiles with two of the reference genotypes (from the 4–8 cm sediment layer). Distinguishing these three experimental genotypes from the reference genotypes under competition proved equivocal using the PGM (EC 5.4.2.2) locus. Therefore, these two genotypes were excluded from analysis involving competitive ability. By identifying the frequency of each experimental genotype relative to the reference genotypes, we compared the competitive abilities of the experimental genotypes under varying P-supply treatments. Experimental genotype frequencies were calculated by dividing the number of individuals of the target genotype by the total number of individuals  at the conclusion of the experiment. Estimated density values were calculated from the fraction of individuals of the target genotype (out of the 12 sampled) multiplied by the total number of individuals in each microcosm following the 21-day period. Estimated density values are represented in Six neonates (less than 24 h old) from each genotype were placed in competition with six neonates of a reference genotype. Three reference genotypes were chosen from the 4–8 cm sediment layer , growth rate, competitive ability, we used separate general linear mixed models (GLMMs) with Daphnia genotype as a random effect. All non-normal data were log-transformed for analyses to fit assumptions for GLMMs. Principal component analysis (PCA) was implemented as a dimension reduction technique to visualize our multivariate data . The PCA was run on a correlation matrix. Correlation matrix PCAs centre and standardize the variables so variables with large values and thus higher chance of variation do not have outsized influences on multivariate patterns. To improve interpretability of the components extracted by the PCA, we applied an orthogonal rotation (varimax) to the components, reporting PC axes with an eigenvalue greater than one. For all traits measured, we did not have an equal number of replicates; therefore, we excluded missing pairwise values for the multivariate analysis . PC axes scores were plotted to visualize the general relationships between all traits measured in a two-dimensional space. Linear regressions and analyses of covariance (ANCOVA) were run on P content, growth rate and competitive ability to test predictions based on ES, i.e. that P content and growth rate should be positively correlated, that growth rate predicts competitive ability, and that P content predicts competitive ability. All tests were performed using SPSS (IBM Statistics v. 22).To test the interactive effects of Daphnia genotypes. All model comparison data analyses were conducted using the R statistical package MuMIn  to measure the relative performance of the models according to Akaike information criterion corrected for small sample sizes (AICc). Daphnia P-use variables were included in the full model as potential predictors, with the model comparison approach used to determine their relative importance to growth and competitive ability. These models were analysed using model selection based on AIC model selection approach to determine which traits best explained growth rate and competitive ability. Using the ΔAICc scores and AICc weights, the top candidate models were identified by removing models with ΔAICc scores greater than 6. Generalized linear models were used to compare a set number of models to the data, measuring the relative support the data gave to each model.A model comparison approach was used to evaluate competing models to explain growth rate and competitive ability in the 3.3.1.D. pulicaria genotypes showed varied responses to P supply conditions with a significant genotype × P treatment interaction. There was a significant genotype-by-treatment interaction for Daphnia %P (p = 0.008), P acquisition, retention and assimilation (p < 0.0001), and P net incorporation (p = 0.001) (Reaction norm plots  of the D= 0.001) .p < 0.0001; figure 1g and p = 0.018; figure 1f and figure 1h and Juvenile growth rate showed a significant genotype by treatment interaction . ANCOVA results revealed that P content and competitive ability did not covary (p = 0.146). P content and competitive ability were not significantly correlated, when separated by treatment . The growth rate hypothesis predicts that rapid growth should be correlated with high somatic P content [p = 0.144; R2 = 0.041; figure 2b). ANCOVA results showed that P content and growth rate covaried (p = 0.002). Faster growth rate predicts higher competitive ability due to fitness advantages related to size. Linear regressions revealed that competitive ability was significantly correlated with growth rate . Growth rate and competitive ability were not significantly correlated when separating by treatment . P content was not correlated with P acquisition  nor with P net incorporation  or P retention . P content was significantly correlated with P assimilation .Stoichiometric models predict that individuals with lower somatic P content will be competitively superior under LP conditions . Competia). Genotypes increased along PC1 to varying degrees in response to LP conditions, thus, representing an increase in P acquisition and assimilation as a response to limited P-supply conditions. Notably, clone 36x did not differ in growth rate between P supply treatments, as it increased its P acquisition and assimilation under LP (figure 4a).The individual loadings on the first three axes of the principal component analysis (PCA) of each dataset, summary of variance explained by the PCA, and eigenvalues for each axis can be found in electronic supplementary material, table S2. Seven of the 10 genotypes are reported in the PCA; one genotype had incomplete P content data and two remaining genotypes shared the same GPI electromorph profiles with two of the reference genotypes. As previously stated, distinguishing these two experimental genotypes from the reference genotypes proved equivocal using the PGM (EC 5.4.2.2) locus and, therefore, these two genotypes were excluded from the multivariate analysis. Percentage of the total variation explained by the first three axes of the PCA was approximately 74%. P-use traits explained the most variation on PC1, explaining approximately 34% of the total variation; PC2 explaining approximately 26% and PC3 explaining approximately 15% . Plottinfigure 43.3.The best supported model for competitive ability included P assimilation and P retention . The bes4.D. pulicaria. Genotypes varied in plastic responses in P content and P kinetics to contrasting P supply conditions, differing in their nature and magnitude of plasticity (a–e). Under P limitation (i.e. LP conditions), P acquisition and assimilation increased, and exhibited more variation among genotypes when compared with HP conditions . P net incorporation and P retention varied significantly between HP and LP treatments . Although growth rates and competitive abilities varied among genotypes (g), these differences were not correlated with P content or P kinetics (b).The results from this study detail how P acquisition, assimilation and excretion (i.e. P kinetics) explain variation in growth rate and competitive ability when compared with P content alone in a population of asticity a–e. Undenditions b,c. P neeatments d,e. Althenotypes g, these kinetics . P contekinetics . Genotypkinetics . P-kinetkinetics b.b). This suggests that under stressful, LP conditions, trade-offs between traits that require a sufficient amount of P are manifested. Large interspecific differences in P content are perhaps important in generating the robust growth–phosphorus couplings [While most genotypes grew faster under HP conditions, this did not correspond to higher P content. We predicted that genotypes with lower P content would exhibit slower growth and be successful under LP conditions. Under LP conditions, some genotypes had an increase in P-use traits, likely to compensate for P deficiencies; i.e. demand of P for RNA biogenesis under the limiting conditions. Additionally, it may be possible that some genotypes can create more biomass with less P body content , e.g. . There wfigure 1ouplings . It is wouplings . At the ouplings –26 shoulp < 0.0001; R2 = 0.27; figure 2c), consistent with previous studies (e.g. [Daphnia genotypes. The reference genotypes (from the 4–8 cm sediment layer) always outcompeted the experimental genotypes (20–24 cm sediment layer) in our study, a result that has been demonstrated previously in D. pulicaria genotypes [Daphnia genotypes depending on nutrient supply stoichiometry [et al. [Daphnia pulex genotypes dominate under certain P supply conditions (C : P ∼ 100) versus others (C : P ∼ 800), demonstrating the large effect P supply can have on competitive outcomes in Daphnia populations. However, such competitive outcomes were probably related to other traits not directly related to P content [In addition to variable strategies to achieve similar growth values, genotypes appear to use different strategies to achieve similar competitive outcomes against a set of common reference genotypes. Faster-growing individuals were found at higher frequencies in the competition experiment (es (e.g. ). While enotypes . Howeverhiometry ,34. Weid content . It is lDaphnia exoskeleton  are better at predicting growth rates and competitive abilities than P content alone tables  and 3. Iarapace; ) should arapace; , thus imarapace; . In addiarapace; , address


In situ sorbent amendment for persistent organic pollutant sequestration in sediment has over the past 15 years steadily progressed from bench‐scale trials to full‐scale remediation applications. Hindering a wider technology uptake are, however, concerns about ecotoxic side‐effects of the most commonly used sorbent, activated carbon, on sensitive, sediment dwelling organisms like Lumbriculus variegatus. Using River Tyne sediment polluted with polycyclic aromatic hydrocarbons (PAHs) and L. variegatus as a case study, sorbent alternatives and magnetic sorbent‐recovery were investigated as potential engineering strategies to mitigate such ecotoxic side‐effects. The potential benefits of contacting the treated sediment with fresh River Tyne water, as would naturally occur over time in the intended applications, were studied.PAH sorbent with less ecotoxic side‐effects than magnetic activated carbon. After 85.1–100% magnetic recovery of this biochar, no ecotoxic side‐effects on L. variegatus were measurable in the treated sediment. Results show that ecotoxic effects of magnetic activated carbon can be alleviated through sorbent recovery. In contrast, contacting treated sediment repeatedly with River Tyne water had no measurable benefits.Magnetic biochar was identified as an effective PAH polluted sediment. © 2017 The Authors. Journal of Chemical Technology & Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.Magnetic biochar is a promising sorbent material for the remediation of  The benefits of hydrophobic organic contaminant (HOC) sequestration in sediment through the addition of a few percent by weight of activated carbon (AC) have been demonstrated in numerous laboratoryLumbriculus variegatus was discovered to be a highly sensitive species negatively affected by AC.L. variegatus gut wall microvilli layer have been reported,While organism survival in AC amended sediments is generally high, and several authors have reported only mild or no activated carbon amendment effects on biota,et al.Asellus aquaticus, and no effect on the survival of L. variegatus, but no reduction in PCB mass fluxes from the sediment to overlaying water. For the use of magnetic activated carbon (MAC), we demonstrated a lasting 98% reduction in PAH sediment pore water concentrations before and after MAC recovery in laboratory experiments, but due to the relatively large, 8.1%, initial MAC dose used in that study and the nonlinearity of the ecotoxic response,L. variegates.Kupryianchyk In the current study we therefore used a lower initial magnetic sorbent dose of 2.5% to sensitively test two complementary hypotheses about the best strategies for minimizing ecotoxic side‐effects of sorbent‐based sediment remediation: (i) effects can be minimized by using alternative sorbent materials; and (ii) effects can be minimized by effective sorbent recovery. Also, we investigated if re‐equilibrating the treated sediment with natural water to replenish contents bound by the sorbents can reduce ecotoxic effects.Superficial sediment was obtained from the intertidal zone of the River Tyne, at Gateshead, Newcastle upon Tyne, UK. The native organic carbon content was 3.0 ± 0.24%. The sediment polycyclic aromatic hydrocarbons (PAHs) were extracted by accelerated solvent extraction using hexane:acetone 50:50 v:v to determine total PAH concentrations by gas chromatography–mass spectrometry. All solvents used were pesticide residue grade, obtained from Sigma‐Aldrich, St. Louis, USA. Available PAH concentrations were determined by using polyethylene (PE) passive samplers.A high quality AC produced from anthracite coal (75–300 µm type TOG) for point‐of‐use drinking water treatment, also used in previous sediment amendment studies,et al.Eight aliquots of 283.7 g (120.0 g dry weight) River Tyne sediment were put into wide mouth amber glass jars, and 3.00 g MagTOG or MagBio  were added into three of the jars, respectively. The two remaining jars without magnetic carbon amendment were used as controls. Three pre‐cleaned PE samplers were put into each jar, and the bottles were sealed with PTFE lined caps and shaken horizontally at 100 rpm. After 3 months, all the PE samplers were recovered, wiped cleaned, and extracted twice for 24 h with 10 mL hexane:acetone 80:20 v:v. The combined extracts were analyzed by GC‐MS as described in Han The iron content of the recovered magnetic materials was measured by digesting 0.5 g of MagTOG or MagBio or 0.36 g of recovered sediment magnetic material in 90 mL of deionized water with 6 mL of concentrated nitric acid and 6 mL of concentrated hydrochloric acid, sonicating for 20 min and then shaking for 5 days. The digestates were diluted 1:3 with distilled water for ICP‐OES analysis on a Varian Vista MPX axial ICP‐OES with CCD, operated according to standard methods for examination of water and wastewater.L. variegatus. Quadruplicate microcosms were set up in 200 mL glass jars filled with 50 g (wet weight) of sediments and 130 mL of overlying artificial freshwater. The starting mass of L. Variegatus was 6.3 ± 1.6 mg fresh weight per worm, the initial dry weight was 17.7% of the fresh weight, and 10 worms were added to each microcosm. The worm dry weight to sediment organic carbon ratio was < 2%. Wet biomass weight, dry biomass weight and reproduction of L. variegatus were used as sensitive endpoints to examine the ecological side‐effects of treatments. A detail method description can be found in Nybom et al.Control, Control‐RW, MagTOG, MagTOG‐R, MagTOG‐RW, MagBio, MagBio‐R, and MagBio‐RW sediment samples were used in ecotoxicity tests with −1 for the 16 US EPA PAHs, which was lower than in the sediment collected from the same location in March 2013 for a previous study (16.08 ± 0.60 µg g−1),−1, respectively .The solid phase PAH concentration in the River Tyne sediment was 6.05 ± 0.42 µg gP = 0.26), but the recovered MagBio had significantly lower elemental iron content than the original MagBio , suggesting partial dissolution of the magnetite particle impregnation in the sediment. The substantial reduction in elemental iron content of recovered MagBio particles, and perhaps also the unrecovered MagTOG particles, might result from the anaerobic conditions in the fine‐grained River Tyne sediment, but exact mechanisms need to be confirmed in a follow‐on study. For the purpose of this study, the main consideration is that MagTOG residuals in sediment following the magnetic recovery are much higher than MagBio residuals .After 3 months of sediment amendment, the recovery rate of MagTOG was only 25.9–36.9%, while the recovery rate of MagBio was 85.1–100% . The wet weight (ww) growth of L. variegatus was, on the other hand, significantly inhibited by an amendment of 2.5% MagTOG, and also by the 1.6–1.9% unrecovered MagTOG residuals . MagBio treatments had no statistically significant effects on L. variegatus ww growth . The dry weight (dw) growth of L. variegatus was significantly inhibited by MagTOG and MagTOG‐RW treatments . The negative dw growth is probably due to a reduction in L. variegatus storage lipid contents often observed in ecotoxicity tests with natural sediments.L. variegatus dw growth .L. variegates.L. variegatus ww of the 2.5% MagBio amendment, all of it in the ingestible particle range (<64 µm), compared with the 2.5% MagTOG amendment, shows that the biochar matrix has lesser ecotoxicity than the activated carbon matrix , since based on the particle size only, one would have anticipated greater ecotoxicity for the finer sized sorbent material. In summary, these observations demonstrate significant scope for optimizing the choice of the sorbent amendment material, and its particle size, through combined consideration of treatment effectiveness ,L. variegatus) in MagBio‐R were statistically indistinguishable from the control . The 36.9% recovery of MagTOG significantly alleviated the inhibition of ww growth and dw growth of L. variegatus by 2.5% MagTOG . There was also an apparent, although not statistically significant, benefit of the MagBio‐R recovery on the dw growth of L. variegatus . These observations demonstrate the benefits of sorbent recovery. It would appear that the sorbent amendment itself, rather than the binding of nutritious content from sediment, caused ecotoxic effects in this study, since adsorbed sediment components would have been removed together with the recovered sorbents, leaving behind a depleted sediment, which would still cause ecotoxic effects. However, nutrient availability was not directly assessed in this study. With the methods employed it can also not be excluded that the observed reduced growth effects occur due to the binding of nutritious contents by the sorbents during sediment digestion, i.e. happening within the gut of the worms, rather than externally. Also, repeated contact of the sediment with large volumes of River Tyne water did not alleviate ecotoxic effects in any of the treatments, which indicates that re‐equilibration with natural water cannot rapidly compensate for the unwanted side‐effects of the sorbent amendment. For MagTOG, the recovery rate from the MagTOG‐RW batch was lower (25.9%) than that of the MagTOG‐R batch (36.9%), and this may explain an apparent adverse ecotoxic effect of the subsequent contact with River Tyne water in this treatment.Following the 85.1–100% magnetic recovery of MagBio, all of the three ecotoxicity assessments (reproduction, ww and dw growth of L. variegatus, replacing AC with biochar as the carbonaceous sorbent matrix, and the recovery of magnetic sorbents, all these measures, to a variable extent, alleviated or annulled ecotoxic amendment effects to the sediment worm L. variegatus, which had sensitively responded to even low AC amendment doses in earlier studies with cleanCompromises can be found between maximizing the treatment benefits of sorbent amendments and minimizing unwanted ecotoxic side‐effects: reducing the magnetic sorbent dose from 8.1% in the previous study


T. cucumerina. Water extracts of freeze dried flowers, fruits, and leaves of T. cucumerina were evaluated for their total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, α-amylase inhibitory activity, and fiber and mineral contents. Antioxidant activity, TPC, and TFC were significantly higher (P ≤ 0.05) in leaves than in flowers and fruits. A significant linear correlation was observed between the TPC, TFC, and antioxidant activities of plant extracts. Although, leaves and flower samples showed a significantly higher (P ≤ 0.05) amylase inhibitory activity than the fruit samples, the overall amylase inhibition was low in all three parts of T. cucumerina. Soluble and insoluble dietary fiber contents were significantly higher (P ≤ 0.05) in fruits than in flowers and leaves. Ca and K contents were significantly higher (P ≤ 0.05) in leaf followed by fruit and flower and Mg, Fe, and Zn contents were significantly higher (P ≤ 0.05) in leaves followed by flowers and fruits. In conclusion, T. cucumerina can be considered as a nourishing food commodity which possesses high nutritional and functional benefits for human health. The present investigation was carried out to determine the nutritional and functional properties of Trichosanthes cucumerina is a well-known plant, the fruit of which is mainly consumed as a vegetable. The plant is commonly called snake gourd, viper gourd, snake tomato, or long tomato in many countries. It is an annual climber belonging to the family Cucurbitaceae and commonly grown in Asian countries including Sri Lanka, India, Malaysia, Peninsula, and Philippines [lippines . The frulippines , 3. The lippines . T. cucumerina was used for treating headache, alopecia, fever, abdominal tumors, bilious, boils, acute colic diarrhea, haematuria, and skin allergy. It has a prominent place in medicinal systems like Ayurveda and Siddha [ T. cucumerina that are used in the traditional medicinal system for treating disease conditions. T. cucumerina is one of the major ingredients in several polyherbal preparations that are prescribed in Sri Lanka for the control of Diabetes Mellitus [ T. cucumerina was found to significantly reduce the blood glucose levels and improve the glucose tolerance of normoglycemic and STZ-induced diabetic rats. In addition to this, Arawwawala et al. have done several studies on Sri Lankan T. cucumerina and have reported it to show antioxidant [ T. cucumerina. Therefore, the present study was conducted to determine the antioxidant potential, total phenolic and total flavonoid content, α-amylase inhibitory activity, soluble and insoluble dietary fiber content, and the essential mineral element content of T. cucumerina grown in Sri Lanka.In ancient medicined Siddha . The whod Siddha , 2, 5–7.Mellitus , 6. In aMellitus , the hotioxidant , anti-inioxidant , antimicioxidant , and gasioxidant  propertiFresh snake gourd fruits, leaves, and flowers belonging to the variety TA-2 were collected from Horticultural Crop Research and Development Institute (HORDI), Gannoruwa, Sri Lanka, in September 2014. Fruits and leaves were harvested three months after cultivation while fruits were collected two weeks after fruit set. All the chemicals and solvents used were of analytical grade.Nearly 50 g of fresh and disease-free snake gourd leaves, flowers, and fruits were washed with distilled water and sliced. The pieces were freeze-dried, ground into fine powder, and stored at −20°C till further analysis. Freeze dried samples (0.1 g) were homogenized at 5000 rpm for 10 minutes in 7.5 mL of deionized water. The homogenized plant samples were centrifuged at 7000 rpm for 15 minutes in a microcentrifuge . The supernatant was filtered through Whatman® 42 filter paper. The extracts were appropriately diluted with distilled water and used for analysis.μL of sample extract, 105 μL of 10% Folin-Ciocalteu's reagent dissolved in deionized water, 80 μL of sodium carbonate , and 15 μL of deionized water. After 3 minutes, Na2CO3 was added and the mixture was incubated for 30 minutes at room temperature. The absorbance was taken using UV-Visible microplate spectrophotometer  at 760 nm. Results were expressed in terms of milligrams of Gallic acid equivalents (mg of GAE) per gram dry weight (g DW). All tests were conducted in triplicate.The total phenolic content (TPC) of the extracts was determined colorimetrically as described by Samatha et al.  with sli2, 5%, w/v) and allowed to stand for 6 min. Afterward, 0.15 mL of aluminum trichloride  was added and incubated for 6 min, followed by the addition of 2 mL of sodium hydroxide , and the volume was made up to 5 mL with distilled water. After 15 min of incubation, absorbance was measured at 510 nm using UV-Visible microplate spectrophotometer. Results were expressed in terms of mg of catechin equivalents (CE) per gram of extract.The procedure described by Agbo et al.  was follμL) was added to different volumes  of aqueous sample extract and diluted with distilled water until the volume reached 250 μL and was allowed to stand for 30 minutes in dark at room temperature. The absorbance was read at 517 nm. The sample concentration providing 50% inhibition (IC50) was calculated.The DPPH assay was performed according to the method described by Sanjeevkumar et al.  with modμL was mixed with 150 μL of ABTS radical solution and absorbance was read over 6 minutes at 1-minute interval at 734 nm. The radical scavenging activity after lapse of 6 minutes was calculated as percentage of ABTS discolouration [The total antioxidant capacity of the extracts was determined using ABTS radical. The ABTS radical was generated by reacting ABTS (1.25 mM) with potassium persulphate (PP) (2.0 mM), which acts as the radical generator. From each extract 50 3·6H2O solution in a ratio of 10 : 1 : 1, respectively. FRAP reagent (150 μL) was added to 100 μL of sample extract and the reaction mixture was incubated at 37°C for 30 min. The absorbance was measured at 593 nm. Fresh working solutions of FeSO4 were used for the standard curve. The results were expressed as mM Fe2+ equivalents per gram of sample.The ability to reduce ferric ions was measured using the method described by Shukla et al.  with somFreeze dried snake gourd sample (0.1 g) was homogenized at 5,000 rpm for 15 minutes in 7.5 mL of 1% Dimethyl Sulphoxide (DMSO). The homogenized plant samples were centrifuged at 7,000 rpm for 15 minutes in a microcentrifuge. The supernatant was used for enzyme assays.μL of α-amylase from porcine pancreas (A3176 SIGMA) in phosphate buffer (PBS)  and 50 μL plant extracts in different concentrations ranging from 5 to 25 mg/mL. The content was incubated for 30 minutes at room temperature followed by the addition of 100 μL of 1% soluble starch (S2004 SIGMA) and incubated for another 3 minutes at room temperature. The reaction was terminated by adding 100 μL DNSA color reagent and placed in 85°C water bath for 15 minutes. After cooling, the sample mixture was diluted with 900 μL of distilled water and absorbance was measured at 540 nm. The control samples were prepared without plant extracts. The % inhibition was calculated and the results were expressed in terms of IC50 value. Acarbose was used as the standard inhibitor.The assay was performed according to Sudha et al.  with sliμL of PBS, 40 μL of plant extracts in different concentrations (5–25 mg/mL), and 40 μL of enzyme in PBS into microplate wells. After 10 minutes of incubation at 37°C, 40 μL of 0.3% soluble starch solution was added and again incubated for another 15 minutes at 37°C. To stop the reaction 20 μL of 1 M HCl was added, followed by the addition of 100 μL of iodine solution (5 mM I2 and 5 mM KI). Immediately after addition the absorbance was taken at 620 nm.Starch-iodine assay was carried out as described by Sudha et al.  with sliμL of PBS, 40 μL of enzyme solution, and 40 μL of plant inhibitor solution. The reaction mixture was incubated for 10 minutes at 37°C and thereafter 100 μL of 1% starch solution was added and the mixture was again incubated for another 15 minutes at 37°C. The turbidity change was monitored at 660 nm. The % inhibition was calculated.The inhibitory activities of plant extracts were quantified based on turbidity measurements according to previous work done by Liu et al.  with sli3 (69%) and 1 mL of H2O2 (30%) in a microwave digestion system and diluted to 50 mL with deionized distilled water. A blank digest was carried out in the same way. The digestion condition in microwave digestion unit was programmed as 15 min ramping time, 10 min holding time or digestion time at 180°C, and 15 min cooling time. Calibration curves were prepared for each mineral using 1000 ppm stock solutions of relevant standard . Necessary dilutions were done in order to get absorbance values. Mineral concentration was given as mg/100 g fresh weight (FW).This analysis was performed according to the method described by Negi et al.  with sliα-amylase (A3306 Sigma) , protease (P3910 Sigma) , and amyloglucosidase (A9913 Sigma)  to remove protein and starch. Enzyme digestates were filtered through glass fritted crucibles. Crucibles containing insoluble dietary fiber were rinsed with dilute ethanol followed by acetone and dried overnight in a 105°C oven and weighed to nearest 0.1 mg. Filtrates plus washings were mixed with 4 volumes of 95% ethanol to precipitate materials that were soluble in the digestates. After 1 h, precipitates were filtered through fritted crucibles. One of each set of duplicate insoluble fiber residues and soluble fiber residues was ashed in a muffle furnace at 525°C for 5 h. Another set of residues were used to determine protein as Kjeldahl nitrogen. Soluble or insoluble dietary fiber residues were obtained through calculation. Total dietary fiber was calculated as the sum of soluble and insoluble dietary fiber.This analysis was performed according to the method described by Shin  with minP values of ≤0.05 were considered to be significant.Data were analyzed using the SAS statistical software version 9.1.3 . Results were calculated and expressed as mean ± standard deviation (SD) of 3 independent analyses.  T. cucumerina aerial parts analyzed in the present study. The differences between the extracts for total phenolic content were significant (P ≤ 0.05). Leaf samples of T. cucumerina showed the highest phenolic content whereas the fruit samples showed the least. As in TPC values, TFC values also differ in the same manner in aerial parts of T. cucumerina  among all three samples. Fruit extract showed the least significant antioxidant activity (P ≤ 0.05) by giving the highest IC50 value as 10.83 ± 0.7 mg/mL. According to the ABTS assay results (P ≤ 0.05) antioxidant activity followed by the flower and fruit extract  mineral element followed by calcium and magnesium. Iron and zinc were recorded as the least. Among all three parts, flower samples had the highest mineral element content while the lowest was recorded in the fruit samples, except for calcium and potassium (P ≤ 0.05). On the contrary, fruit samples were significantly high in potassium (P ≤ 0.05) (Potassium was recorded as the highest ( ≤ 0.05) .Insoluble dietary fiber (IDF) content of all three parts was higher than soluble dietary fiber (SDF). When considering the total dietary fiber (TDF) content, fruit samples had significantly higher fiber content (31.76 ± 3.7 g/100 g) followed by flowers and leaves  . The sta T. cucumerina possess antioxidative properties. These results are in agreement with those reported by Choudhary et al. [ T. cucumerina. According to Ademosun et al. [ T. cucumerina was significantly (P ≤ 0.05) higher than that of a tomato variety of Lycopersicon esculentum Mill. var. esculentum.Snake gourd contains a rich variety of nutrients, vitamins, and minerals that are essential for human health, including significant levels of dietary fiber, a small amount of calories, and high levels of protein. These medicinal plants are reported to possess different pharmacological properties due to the presence of phytochemicals such as alkaloids, flavonoids, glycosides, tannins, and steroids. It is well-known that antioxidative properties of plants help to protect the body from free radical damage in cell structures, nucleic acids, lipids, proteins, and other body components and thereby reduce the risk and progression of many acute and chronic diseases like cancer, cardiovascular diseases, diabetes, and other metabolic syndromes . The resy et al.  in Indian et al. , the tot50 values for leaves and fruit as 278.29 ± 2.98 μg/mL and 260.30 ± 2.23 μg/mL, respectively. The antioxidant activities of T. cucumerina well correlated with the amount of total phenolic and flavonoid contents. Several reports have shown a close relationship between total phenolic content and antioxidant activities [Antioxidants are substances that have the ability to neutralize free radicals. In a normal healthy human body, the scavenging of prooxidants such as reactive oxygen species (ROS) and reactive nitrogen species (RNS) is effectively regulated by various antioxidant defense systems. With the exposure to adverse physicochemical, environmental, or pathological conditions, the regularly maintained balance is shifted in favor of prooxidants. This results in oxidative stress which is highly responsible for adverse health conditions including autoimmune destruction in the human body . Hence, tivities .α-amylase activity [ T. cucumerina, the findings of Ademosun et al. [α-amylase activity with an IC50 value of 2.15 ± 0.09 mg/mL.Natural plants or their products are related in retarding the absorption of glucose by inhibiting starch hydrolyzing enzymes, such as pancreatic amylase and glucosidase. The inhibition of these enzymes delays carbohydrate digestion thereby reducing the glucose releasing rate and consequently lowers the increase in postprandial plasma glucose. Several indigenous medicinal plants have a high potential in inhibiting activity . Though n et al.  have sho T. cucumerina. According to Ilelaboye and Pikuda [ T. cucumerina seeds contain iron (187 mg/kg DM), zinc (37.5 mg/kg DM), calcium (1643 mg/kg DM), magnesium (1896 mg/kg DM), and potassium (8704 mg/kg DM).Dietary minerals and trace elements are chemical substances required by living organisms in addition to carbon, hydrogen, nitrogen, and oxygen that are present in nearly all organic molecules. These elements perform various functions, including building of bones and cell structures, regulating the body's pH, carrying charge, and driving chemical reactions. Minerals and trace elements are usually obtained through the diet. Though food sources of animal origin provide most essential minerals, some plants are also considered as rich sources of minerals. Several other researchers have reported positive results for mineral element analysis ofd Pikuda , T. cucu T. cucumerina leaves as 12 g/100 g similar to that of the present study. However, Hettiaratchi et al. [ T. cucumerina fresh fruit into a salad meal did not increase the total fiber content in comparison to the control meal incorporated with Lasia spinosa rhizome.Most of the foods with a low or medium glycemic index are reported to contain considerable amounts of dietary fiber , 29. Thei et al.  had repo T. cucumerina is rich in phenolic compounds and has good antioxidant activity. The plant can also be considered as a good source of essential mineral elements and rich in both soluble and insoluble dietary fiber. Several studies have also proven that T. cucumerina possesses so many functional health benefits [ T. cucumerina can be considered as a natural source of functional nutrients which may yield many beneficial effects to the human body.The results of this study suggest thatbenefits –11. Thus Trichosanthes cucumerina, fruits, leaves, and flowers possessed significant amounts of beneficial compounds. The highest concentration of phenolic compounds, flavonoids, and antioxidant capacity was found in the leaves and least in the fruits. The α-amylase inhibitory activity of the plant was not significant. Furthermore, the mineral element content of the leaves and flowers is considerably higher than that of the fruit, while the fiber content is significantly high in the fruit compared to the leaves and flowers.From the present study it is concluded that the water extract of


Six catheter instillations at weekly intervals were used. Cystoscopy and biopsy were performed before and 3 months after the start of treatment. Suramin was assayed using high-performance liquid chromatography, vascular endothelial growth factor (VEGF) using ELISA (enzyme-linked immunosorbent assay), and urinary protein profile using surface-enhanced laser desorption ionisation mass spectroscopy (SELDI). Minimal systemic absorption of Suramin was found at the highest dose of 150 mg ml−1. Urinary VEGF was affected by Suramin at doses above 50 mg ml−1, corresponding to the estimated threshold of saturation of Suramin binding to urine albumin. SELDI showed a specific disappearance of urinary protein peaks during treatment. Intravesical Suramin shows lack of toxicity and low systemic absorption. The results of this phase I trial support expanded clinical trials of efficacy at a dose of 100 mg ml−1 intravesically.Suramin is an antitrypanosomal agent with antineoplastic activity, but with serious systemic side effects. We administered Suramin intravesically to determine a concentration with low toxicity but with evidence of a pharmacodynamic effect, to recommend a dose level for phase II trials. This was an open-labelled, nonrandomised dose-escalation phase I study. In all, 12 patients with a history of recurrent superficial bladder cancer were grouped into four dose levels (10–150 mg ml Bladder cancer has an incidence of 30/100 000 per year and three-quarters of patients present with superficial disease. In all, 30% of intermediate-risk superficial bladder tumours will recur within 2 years despite intravesical treatment with mitomycin C, and around 6% may progress to cause death within a median follow-up of 7 years  , systemic absorption should be very low. Additionally, in the plasma, 99.7% of Suramin is protein bound, which may interfere with its availability to bind growth factors. The much lower protein levels in urine could mean greater bioavailability. Graham reported excellent efficacy when given intravesically to rats in a chemically induced model of bladder cancer .in situ (from four random bladder biopsies taken at pretreatment cystoscopy) were excluded. Concurrent treatment for other malignancies, prior radiotherapy, anticoagulant therapy, a history of adrenal insufficiency or steroid therapy were also exclusion criteria, as well as clinically significant hepatic or renal disease.Patients for whom a rigid cystoscopy had been indicated following previous treatment for superficial bladder cancer or who had symptoms indicating a possible recurrence were included. Recurrences were detected using flexible cystoscopy. Entry criteria were that patients had to be 18 years or older, have a World Health Organization (WHO) performance status of 0–2, and have no toxic manifestations of previous treatments. Inclusion criteria were presence of Ta, T1, and Grade 1 or 2 multiple tumours (but not >7). Patients with single or >7 tumours, stage T2−4b, Grade 3 or carcinoma Suramin was supplied by the NCI (USA), Division of Cancer Treatment and Diagnosis, in lyophilised form as a powder stored at room temperature. Suramin is freely soluble in water. The dose was diluted in 60 ml normal saline for each intravesical instillation. Patients were grouped into four treatment levels, with intergroup dose escalation as the trial progressed. Significant, that is, Grade 3 (NCI Common Toxicity Criteria), toxicity would prevent progression to the next treatment level and expand recruitment to a maximum of six patients at that level. Dose levels are described in The protocol was approved by the CRC (Cancer Research Campaign) Protocol Review Committee, the CIRB , and the Local Research Ethics Committee (LREC). The study was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained. This was an open-labelled, nonrandomised phase I study with an intended recruitment of 12 patients. Pretreatment evaluation included full history, physical examination, and assessment of WHO performance status. In addition, a complete blood count, clotting screen, renal, and liver biochemical profiles and urine analysis were performed. These were repeated weekly and 3 weeks after the end of treatment. At the pretreatment transurethral resection of bladder tumour (TURBT), random biopsies of normal bladder and samples of the tumour were taken, and both were fixed in formalin and paraffin embedded.in situ for 2 h, during which time the patient remained horizontal, and was asked to turn through 90° every 15 min.Patients received their first instillation of Suramin at least 14 days postcystoscopy and TURBT. This was to allow the bladder to heal and to reduce systemic absorption. The drug was then given once a week for 6 weeks. The bladder was drained prior to and postinstillation. Treatments were left At 3 weeks after the end of the treatment, a further cystoscopy was performed under general anaesthetic and further random biopsies taken with tumour resection if necessary.g for 10 min within 30 min of sampling. The plasma was stored in aliquots at −70°C. The volume of urine drained from the bladder at the end of 2 h was measured. An aliquot of 10 ml of urine was collected, 1 ml of protease inhibitor was added , and the urine was centrifuged at 2000 g for 10 min within 30 min of sampling. Aliquots were stored at −70°C. It has previously been shown that protease inhibitor does not affect VEGF results (Blood samples were collected in EDTA tubes and centrifuged at 2000 μl of 1 M tetrabutyl ammonium bromide (ion-pairing agent) and 200 μl 10 μg ml−1 methylphenyl-phenyl-hydantoin  were added to a 250 μl sample (plasma or urine). Proteins were then precipitated with 500 μl methanol by incubation at 4°C for 30 min, after which the supernatant was injected into the HPLC system. Standard curves covered the range of 0–30 μg ml−1 plasma and 0–200 μg ml−1 urine. Urine samples were diluted by up to 900-fold (depending on the dose level) with water to ensure the concentration was within the standard range. Separation of peaks of interest was achieved using a 5 μM Hypersil C18 column  and a mobile phase comprising 10 mM phosphate buffer , with 4 mM tetrabutylammonium bromide and 52.5% methanol. Eluting peaks were monitored at 230 nM by UV detection. The limit of quantitation was 0.5 μg ml−1 plasma and 5 μg ml−1 urine. Reproducibility (covariance) of quality control samples at 0.5, 4, and 20 μg ml−1 plasma and 5 and 50 μg ml−1 urine was <12%. This protocol released Suramin from protein and measured total Suramin.Suramin concentration in plasma and urine was determined using a high-performance liquid chromatography (HPLC) assay following protein precipitation. Briefly, 200 −1.VEGF was measured in the urine and plasma using a standard enzyme-linked immunosorbent assay (ELISA) (R&D Systems) before each Suramin dose and was compared with that detectable after each Suramin dose. This ELISA has previously been tested for use with urinary samples for recovery of VEGF from urine (β-lactaglobulin A (MW 18 363.34). Samples were loaded using a bioprocessor as described previously  NP40 and for the H4 chips it was prewet with 50% acetonitrile with 10% acetonitrile/0.1% TFA being used for wash steps. Additionally, in each case, sample loading was standardised on the basis of creatinine with a diluted volume of urine equivalent to a final creatinine concentration of 2.5 mM being loaded in a 50 ml volume. Samples were analysed using parameters of a high mass of 100 kDa, optimum mass range 3–20 kDa, laser intensity of 210, sensitivity of 10 and collecting 50 transients across the spot surface. This was carried out on samples pre- and post-Suramin dose 1, pre- and postdose 5 or 6, and on the 4 weeks post-treatment samples for each of three patients (patient 7 at dose level 2 and two patients 9 and 12 at dose level 3). Samples were all analysed on the same batch of chips on a single day to minimise variability.To assess the general effect of Suramin instillation on global urine protein profiles, urine samples were also analysed using SELDI  with the SAX2 and H4 chip surfaces. The instrument was calibrated at the start and end of the run using bovine ubiquitin (MW 8564.8), bovine superoxide dismutase (MW 15591.4), and bovine M citrate, pH 6.0. Primary antibody was applied (1 : 4 in 1 × TBS) for 1 h. Control slide used was normal human tonsil where staining of vessels, stromal cells and luminal surface was seen. Labelling was detected using anti-mouse system (Dako) as per the manufacturer's instructions. Sections were then counterstained with haematoxylin and mounted. Staining of blood vessels was performed with anti-CD34 (QBEnd 10 antibody) at a final concentration of 1 : 100 using a similar protocol. The number of CD34-stained microvessels was counted in the lamina propria immediately adjacent to the urothelium on a high-power (× 40 objective) lens and the proportional vascular space volume determined using a Chalkley point graticule  receptor using 11B5 monoclonal antibody .  and one patient developed minor lymphopenia at the lowest dose level, of unproven significance. No serious adverse events were noted and no patients complained of urinary symptoms related to treatment. In fact, two patients previously with nocturnal frequency of micturition noted a marked improvement in this symptom while receiving treatment. There were no drug-related adverse events above Grade 1.vs 1/6 Fisher's exact test not significant P=0.2).Three patients had recurrent tumours and two had dysplasia at the final cystoscopy. Four of these were in the lower two dose groups .All urine samples immediately before preinstillation had undetectable levels of Suramin. Urine drained at the termination of each instillation contained levels as shown in μg ml−1. Two patients at dose level 4 had detectable Suramin levels above 6 μg ml−1. These were in patient 11 after instillations 5 and 6 with levels of 6.3 μg ml−1, 1163 μg ml−1, respectively, and in patient 12 after instillation 5 with a level of 333 μg ml−1. High levels were diluted and rerun, with the repeat confirming the value in both cases. No clinically traumatic catheterisation occurred during the study. These patients were not receiving any different drugs on these days compared to other days.In all, 12 patients were evaluable for pharmacokinetics. All plasma samples for patients up to and including level 3 had no detectable Suramin, that is, less than 0.5–6 −1 causing an approximate 40% fall in the measured level of VEGF at all concentrations, while 150 mg ml−1 caused almost a 100% fall at all concentrations compared with control. A number of means were tried to disrupt the interaction between Suramin and VEGF. These included dialysis through a semipermeable membrane, passage through an ion-exchange column (Suramin having a negative charge), dissolution in 4 M NaCl, and the addition of 10% SDS followed by boiling. None of these methods had any effect on the VEGF–Suramin binding as measured by ELISA (data not shown). A similar effect was demonstrated using spiked human urine samples; thus, in the data that follows, VEGF levels must be interpreted in the light of measured Suramin concentrations and reflect free VEGF concentrations.Suramin shows high affinity for the heparin-binding growth factor VEGF. Standard samples of VEGF were spiked with increasing concentrations of Suramin, with resulting interference of the VEGF ELISA as shown by the poor recoveries in In all, 12 patients were evaluable for pharmacodynamics. Plasma VEGF results did not change significantly over the course of treatment at any dose level. Plasma levels of VEGF were in the normal range for all patients, except patient number seven, who for unknown reasons had persistently raised levels.−1. Suramin did not alter urinary VEGF over the course of treatment on average, as assessed by the baseline value before each instillation.Urinary VEGF was quantified by ELISA with values normalised to urinary creatinine, to adjust for variation in urine concentration . Mean VEAlthough the convention with urine samples is to normalise results for urine creatinine to correct for hydration status, differences in protein loading on SELDI chips can influence the profile . However−3 (s.d. 45) and in post-treatment 140 mm−3 (s.d. 56), and mean proportional vascular volume estimated using the Chalkley random point graticule was 17% (s.d. 5.2) and 18% (s.d. 3.0), respectively (NS paired t-test).In all, 22 normal bladder biopsies from 11 patients pre- and post-treatment were available for staining. Mean microvessel count in pretreatment samples was 141 mmn=4, mean 186 mm−3, s.d. 66) was significantly higher (P=0.04) than in mildly or noninflamed samples  and there was a trend to higher proportional vascular volume in severely inflamed samples . The number of inflamed samples in pre- and post-treatment groups was not significantly different: two severely inflamed samples in each group, overall seven biopsies reported as any degree of inflammation in pretreatment group, and nine inflamed in post-treatment. Mean microvessel count in severely inflamed samples (χ2 test).VEGF staining was scored on the basis of intensity and breadth of staining of the urothelium. Positive was defined as a biopsy showing full thickness epithelial staining either in patches or along the entire epithelial border. All biopsies showed staining of the umbrella layer of the urothelium, but there was no significant difference between pre- and postbiopsy samples in full thickness staining: four of 11 pretreatment biopsies stained positive and two of 11 post-treatment stained positive. Three of nine noninflamed and three of 13 inflamed were positive. Neither difference was significant , −1 (−1. Full inhibition of the VEGF ELISA was only attained in patients treated with level 3 dosing (100 mg ml−1 Suramin instilled) in whom mean postdose urinary Suramin concentration was 29 mg ml−1. Of particular note, this is 300 times higher than the concentration required to inhibit transitional cell carcinoma cell growth in culture , −1 . The one−1 dose in 60 ml of normal saline over a 6-week period  with the majority of the nine patients receiving between 0.3 and 154 mg ml−1 demonstrated high systemic absorption (334 and 1163 μg ml−1 plasma level postdose). There was no associated clinical adverse effect. Plasma levels above 275 μg ml−1 when maintained for over 4 weeks by intravenous infusion cause clinically significant neurological adverse effects such as paresis  after these high levels were detected, suggesting that these represent transient peaks and do not produce a reservoir.In our study, two treatments at the highest dose level of 150 mg mlin vitro and therefore is unlikely to reflect effects of Suramin on release of protein by bladder cells. Suramin, which is negatively charged, may compete with proteins binding to the chip surface with selective effects due to relative binding affinities. Alternatively, binding of Suramin to specific proteins may either alter their binding properties or cause their aggregation.SELDI provides a semiquantitative means of profiling, although many factors may affect the results including ion suppression. The general trend was of less protein peaks in the urine samples post-Suramin, although not all proteins were affected to the same extent. This was also seen if Suramin was spiked into urines Three patients had recurrent tumours and two had dysplasia at the final cystoscopy. Four of these five recurrences were in the lower two dose groups. These numbers are insufficient to reach statistical significance, but the trend to fewer recurrences in the higher dose groups supports the laboratory end point determination.−1. Inhibition of the VEGF ELISA occurred reliably in patients at the 100 mg ml−1 level but not at lower levels. These results support the progression to expanded clinical trials of efficacy using a dose of 100 mg ml−1 intravesically.In conclusion, intravesical Suramin is well tolerated in the treatment of patients with recurrent superficial bladder cancer. Levels are undetectable in the blood until the intravesical dose reaches 150 mg mlin vitro bladder explant model (Our


Vascular endothelial growth factor (VEGF) is a major target for the inhibition of tumour vascularisation and the treatment of human cancer. Many tumours produce large quantities of VEGF, and as a result, diagnosis and prognosis of cancer may be predicted by measuring changes in VEGF concentrations in blood. In blood, the VEGF may be located in the plasma, or in the blood-borne cells and formed elements, in particular, platelets and leukocytes. In this study, we collate the measurements of VEGF in platelets, leukocytes, plasma and serum for breast, prostate, colorectal and other cancers. In addition, we analysed the concentration of VEGF in tumour tissue itself, as well as for other tissues in the human body. Although the concentration of VEGF in tumours is high, the size of tumours is small compared to other tissues, in particular, skeletal muscle. Thus, the total quantity of VEGF in tumours and in blood is small compared to the quantity in muscles. This large reservoir of VEGF may have important implications for the treatment of cancer. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has important roles in growth and development, wound healing and tumorigenesis. The vascular endothelial growth factor (VEGF) family of proteins has a pivotal role in regulating tumour angiogenesis .Vascular endothelial growth factor stimulates cell survival, migration and differentiation. It induces neovascularisation, and is required for the establishment of haematopoiesis; in malignant tumours, VEGF supports development of tumour vessels, which may lead to increased vascular permeability, and is shown to have a correlation with cancer prognosis and diagnosis  were also excluded. Based on these criteria, 12 studies were excluded from our collection . Both plasma and serum samples were subsequently aliquoted and stored at −20 to −80°C before assay.Both plasma and serum have been commonly used to determine VEGF levels in the blood. From our reviewed literature, peripheral venous blood samples were drawn. To prepare plasma, blood samples were put in test tubes with an anticoagulant (either trisodium citrate or ethylenediaminetetraacetic acid). The test tubes were then left for 0–30 min before centrifugation, 1000–3000 × g, for 15–60 min. The resulting supernatant (tumour cytosol) was then stored at −70 to −80°C until analysis.To obtain tumour cytosol measurements, tumour tissues obtained during surgery were immediately frozen in liquid nitrogen. To prepare the samples, the tissue samples were diluted in a buffer. The tissues were then either homogenised with Ultra Terrex, pulverised with microdismembrator or diced with a scalpel. The homogenised tissue samples were then centrifuged at 800–105 000 × Enzyme-linked immunosorbent assay (ELISA) was the adopted assay type for 74% of the studies. Among those using ELISA, 40% obtained the assay kit from R&D Systems, . The remaining 60% used kits from 14 other different companies. Five other different assay types were also used, including quantitative sandwich enzyme immunoassay, enzyme immunoassay, chemiluminescence immunosorbent assay, human VEGF immunoassay quantigo kit and immunofluorometric assay.The weighted average and s.d. of VEGF concentrations were evaluated in all cancer studies for various compartment of the body . Our calculations were based on the following equations: ni: number of subjects reported in each paper; xi, VEGF level reported in each paper.t-test was used. A probability of <0.05 was taken to be significant. The overall statistical meta-analysis is given in −1, pg mg−1 protein, pg 10−6 cells, 106 cells ml−1. Unit conversion was performed whenever necessary. Concentrations are rounded to whole numbers.A two-sample, one-tailed Student's Our literature search identified 64 references containing quantitative information to be included in this analysis, including 19 breast cancer, 13 prostate cancer, 13 colorectal cancer and 19 other cancer studies. Details for each of these studies are given in vs 17–287 pg ml−1). Plasma VEGF levels have a range of 37–310 pg ml−1 (cancer) vs 27–30 pg ml−1 . Tumour cytosol VEGF values range from 140 to 693 pg mg−1 protein, but there is no control equivalent in healthy subjects or other tissues in these studies. We will compare these values with measurements from other tissues reported in other studies.The main results on healthy and cancer VEGF levels are summarised in vs 90–298 pg ml−1 (positive); PgR status: 75–141 (negative) vs 89–187 pg ml−1 (positive)). However, both ER and PgR tumour cytosol values deviate from the above data, where ER-negative values are 1.5–2 times higher than ER-positive values (360–700 vs 180–560 pg mg−1 protein) and PgR-negative values are 1.5 times higher than PgR-positive values (289–760 vs 212–510 pg mg−1 protein).The relationship between VEGF in cancer and expression of hormone receptors for oestrogen and progesterone was also examined. Serum values appear to be slightly higher for both positive ER and PgR status, although the difference does not appear to be significant (ER status: 75–271 (negative) vs 17–171 pg ml−1 ; plasma: 32–730 cancer vs 13–61 pg ml−1 ).Results are summarised in vs 173–391 pg ml−1 ; plasma: 19–211 cancer vs 9–126 pg ml−1 ). Whole blood values are relatively high ), although results may have been skewed with only two studies involved. Tumour cytosol values range from 189 to 984 pg mg−1 protein, which are approximately 1.5 times higher than those in breast cancer studies.Results are summarised in vs 51–318 pg ml−1 ; plasma: 23–137 cancer vs 9–26 pg ml−1 ). For whole blood VEGF, cancer values are again higher when compared to serum and plasma data ).Results are summarised in vs 222 pg ml−1).Results are summarised in To evaluate VEGF distribution in the body, we combined all the studies and derived the average VEGF concentration for serum and plasma. To determine the total quantity of serum or plasma VEGF in the body, we multiplied concentrations by the total volume of serum or plasma, 2.26 l, as explained below  was estimated using a protein mass ratio of 160 mg protein g−1 fat-free tissue and tissue density of 1.06 g tissue ml−1  in these tissues was estimated using a protein mass ratio of 160 mg protein g−1 fat-free tissue and tissue density of 1.06 g tissue ml−1 , the volume of blood (5 l) and the density of blood (1060 kg m−3). Assuming that the body consisted only of tissue and blood, we estimated the total VEGF to be between 318 and 17448 μg.Vascular endothelial growth factor concentration in normal colon tissues and skeletal muscle ranged between 27.3 and 1500 pg mg−1 tissue . This may indicate that VEGF is more readily compartmentalised in blood and muscle. Further research will be needed to determine how VEGF is concentrated and transported in each of the compartments of the body.M , 13.3–39.4 pg ml−1 tissue (breast tumour). Note that the extracellular concentration is similar to the plasma concentration   VEGF in the interstitial space of human breast tumour and muscle vastus lateralis have been measured using microdialysis. For both tissues, this concentration is in the range of 0.5–1.5 pntration , suggestr-bound) . Thus, tM in acute leukaemia patients  (−1 in cancer patients (of various types of cancer)   than in healthy subjects (cancer: 2929 pg mlVascular endothelial growth factor secreted from various tissues will eventually be cleared by the kidney. Urine VEGF values have been reported to be higher in cancer patients when compared to healthy controls (Our results provide an integrative analysis of VEGF levels in various compartments of the body in cancer patients and address important issues in VEGF data interpretation. Conclusions drawn from VEGF values in different cancer types were consistent. Within the blood compartment, VEGF is mostly concentrated in the platelets, although a significant portion was localised in leukocytes during cancer development. Large quantities of VEGF were reported in tumour and skeletal muscle, the latter of which suggested an intracellular VEGF source.The most surprising result of this analysis is that even in cancer, tumours are not the largest source of VEGF in the body. Other tissues, and in particular skeletal muscle, appear to contain a large reservoir of VEGF, and this should be a consideration in the design of cancer therapeutics. For example, the systemic administration of anti-VEGF antibodies may have to overcome the effects of a large non-tumour-derived VEGF reservoir.


Therefore, plasma may be preferred for vascular endothelial growth factor measurements. However, which specimen is the best predictor of survival is still debated. The present study analyzed the prognostic value of matched preoperative serum and plasma vascular endothelial growth factor concentrations in patients with colorectal cancer. To establish the reference range among healthy people, vascular endothelial growth factor was analyzed in 50 matched EDTA-plasma and serum samples from healthy blood donors. Preoperatively, in 524 patients with colorectal cancer, matched plasma and serum vascular endothelial growth factor concentrations were analyzed. In the colorectal cancer patients, the median plasma vascular endothelial growth factor concentration (44 pg ml−1) was significantly (P=0.01) higher than the median plasma vascular endothelial growth factor concentration (30 pg ml−1) in the healthy blood donors. In serum, no significant (P=0.30) difference in the median vascular endothelial growth factor concentration was found between colorectal cancer patients (268 pg ml−1) and healthy blood donors (220 pg ml−1). The preoperative vascular endothelial growth factor concentrations were dichotomized by the 95th percentile of the healthy blood donors . In univariate survival analyses, both high plasma vascular endothelial growth factor (>112 pg ml−1) and high serum vascular endothelial growth factor (>533 pg ml−1) predicted a reduced survival. In multivariate survival analyses, high serum vascular endothelial growth factor (>533 pg ml−1) independently predicted a reduced survival , while high plasma vascular endothelial growth factor (>112 pg ml−1) did not . This study indicates that preoperative serum vascular endothelial growth factor apparently is a better predictor of overall survival than the preoperative plasma vascular endothelial growth factor.In serum, the major part of vascular endothelial growth factor derives from British Journal of Cancer (2002) 86, 417–423. DOI: 10.1038/sj/bjc/6600075www.bjcancer.comThe Cancer Research Campaign© 2002  The median age of the patients at the time of operation was 69 (33–90) years, and 316 men and 208 women were included. All patients had their primary tumours resected and none were given chemotherapy or radiotherapy before or after the operation. All patients had histologically verified carcinoma localized in the colon or in the rectum and were staged according to the Dukes' classification with an added group D that identified patients with solid metastases or disease that had not been completely resected. The distribution of Dukes' stages according to site of tumour is shown in Table 1The peripheral blood samples were collected in endotoxin-free silicone coated tubes  with EDTA as additive (plasma) or without additive (serum), after written informed consent in accordance with the Helsinki II declaration. The plasma samples were centrifuged  immediately after the aspiration, and the plasma was removed and stored at −80°C until analyzed. The serum samples were allowed to clot at room temperature for 30 min before centrifugation  and the serum was removed and stored at −80°C until analyzed. Blood samples were obtained preoperatively from the patients on the day of their operation, just before skin incision. The blood samples from the volunteer blood donors were obtained at the time of their routine donation of blood in the blood bank.Before analysis, the plasma and serum samples were thawed at room temperature. The VEGF concentration was measured with a commercially available human VEGF quantitative enzyme linked immunosorbent assay (ELISA) kit , according to the instructions given by the manufacturer. All analyses were made in duplicate and the mean value was used for statistical calculations.−1 respectively) or otherwise scored as high. In addition, the patients were grouped into three strata by the 10th and 90th or the 25th and 75th percentiles of their plasma and serum VEGF concentrations. The end-point for survival analysis was death of any cause. The Kaplan–Meier method was used to estimate survival probabilities, and the log rank test was used to test for equality of strata. The Cox proportional hazard model was used for multivariate analysis. The assumption of proportional hazards was verified graphically. Rank statistics were used to calculate correlation coefficients and to test hypothesis on location. Tests of independence were done with the χ2 test. McNemars test was used for paired proportions. The level of significance was set at 5%.The SAS® software package  was used to manage patient data and for statistical analysis. In the 524 CRC patients, the plasma and serum VEGF concentrations were scored as low if VEGF was less than or equal to the 95th percentile of the normal controls (112 and 533 pg ml−1 (range 0–369). There was no significant correlation  between age and plasma VEGF among the blood donors and no significant difference in plasma VEGF (P=0.86) between men and women.In the 50 healthy blood donors, the median plasma VEGF concentration was 30 pg ml−1 (range 0–1185). There was no significant correlation  between age and plasma VEGF among the CRC patients, and no significant difference in plasma VEGF (P=0.32) between men and women. The percentile plot of the plasma VEGF measurements in the 524 CRC patients and in the 50 blood donors is shown in Figure 1AP=0.01) higher than the medium value for the healthy blood donors.In the 524 patients with CRC, the median plasma VEGF concentration was 44 pg ml−1 (range 0–440); stage B: 44 pg ml−1 (range 0–1185); stage C: 44 pg ml−1 (range 0–706), and stage D: 74 pg ml−1 (range 0–543). The median plasma VEGF concentration significantly (P=0.001) increased with advanced Dukes stage. Patients with the primary tumour localized in the colon, had significantly (P=0.01) higher median plasma VEGF concentrations than patients with the primary tumour localized in the rectum.Stratified by Dukes stage, the median plasma VEGF concentration among the CRC patients was as follows: Stage A: 27 pg ml−1 (range 46–983). There was no significant correlation  between age and serum VEGF among the healthy donors, and no significant difference in serum VEGF (P=0.74) between men and women was found.In the 50 healthy blood donors, the median serum VEGF concentration was 220 pg ml−1 (range 9–2500). There was no significant correlation  between age and serum VEGF among the patients, and no significant difference in serum VEGF (P=0.22) between men and women. The percentile plot of the serum VEGF measurements in the 524 CRC patients and in the 50 blood donors is shown in −1) was higher than the medium serum VEGF concentration in the healthy controls (220 pg ml−1). However, in contrast to the median plasma VEGF concentration between CRC patients and healthy blood donors, the difference in the median serum VEGF concentration was not statistically significant (P=0.30).In the 524 patients with CRC, the median concentration of serum VEGF was 268 pg ml−1 (range 9–1500); stage B: 266 pg ml−1 (range 15–1975); stage C: 263 pg ml−1 (range 15–2500), and stage D: 304 pg ml−1 (range 19–1475). Patients with Dukes stage D disease had significantly (P=0.001) higher serum VEGF concentrations compared to patients with Dukes stage A, B and C disease, while the latter three groups had comparable concentrations. Patients with the primary tumour localized in the colon, had significantly (P=0.01) higher serum VEGF levels than patients with the primary tumour localized in the rectum.Stratified by Dukes stage, the median serum VEGF concentration among the colorectal cancer patients was as follows: Stage A: 261 pg mls=0.64) between the 524 matched serum and plasma VEGF measurements is shown in Figure 2P=0.01, McNemars test). This analysis indicated that, although serum and plasma concentrations are correlated, grouping by the 95th percentile of normal donors, significantly more positives are identified by plasma VEGFThe correlation (r−1 (n=105) had a reduced overall survival  compared to the patients (n=419) with plasma VEGF concentrations equal to or below this level , (n=81), overall survival was significantly (P=0.006) reduced, compared to patients with VEGF concentrations below or equal to this level (n=443) (−1 (n=71) had significantly (P=0.01) reduced overall survival, compared to colon cancer patients (n=223) with lower VEGF concentrations ,  had significantly (P=0.003) reduced survival compared to the patients (n=237) with low VEGF concentration   . In the P=0.93), . Dichoto (P=0.3) .−1 had a significantly (P<0.0001) reduced survival compared to the patients with lower concentrations. Additionally, CRC patients with preoperative serum VEGF concentrations lower than 76 pg ml−1 had a significantly better prognosis than CRC patients with VEGF concentrations above this level. Using the 25th and the 75th percentiles for stratification shows a less pronounced effect although statistically significant (P=0.02). This effect was not seen with plasma VEGF.In the above-mentioned calculations, the cut-off point was set to the upper limit of the 95th percentile of healthy controls. However, this level may not be the relevant scoring. In order to study if a trend could be detected, strata were defined by the 10th and 90th percentiles of the plasma and serum VEGF concentrations. This division as seen in Figure 6−1) significantly (P=0.015) predicted a reduced overall survival, while high preoperative plasma VEGF level (>112 pg ml−1) did not (P=0.23). In patients with rectal cancer, neither high serum VEGF concentration nor high plasma VEGF concentration independently predicted a reduced overall survival (P=0.19 and 0.41 respectively). Additionally, patients with the tumour located in the rectum had a significantly (P<0.0001) worse prognosis than patients with the primary tumour located in the colon. In Table 3−1 are shown. This survival analysis indicates that the 95th percentile may not be the relevant clinical cut-off point for serum VEGF. Furthermore, the analysis showed that the patients with the highest 10% of the serum VEGF concentrations had a significantly (P=0.0005) reduced survival compared to the CRC patients with lower VEGF concentrations, while the patients with the lowest 10% of the serum VEGF concentrations had a significantly (P=0.04) better prognosis than the patients with higher preoperative serum VEGF concentrations.Multivariate survival analysis was performed including Dukes stage, gender, age, topographical tumour localization, and VEGF level  difference in Dukes stage was found between rectal and colon cancer patients, so that rectal cancer patients in general had a less advanced disease at the time of diagnosis. However, in an analysis including plasma and serum VEGF, topographical tumour localization and Dukes stage, patients with the tumour localized in the colon, independently of Dukes stage, still had significantly higher plasma and serum VEGF levels  than patients with the tumour localized in the rectum. Therefore, the difference in plasma and serum VEGF levels between rectal and colon cancer is not simply a reflection of the difference in distribution of Dukes stage, but seems to be a genuine biological difference between the two tumour types.A significant (in vitro coagulation (in vitro degranulation of platelets, the increased platelet-derived VEGF may influence the biology of a present tumour in vivo, and may presumably reflect tumour burden at the time of surgery.Several studies have indicated that serum is unsuitable for VEGF measurements since the significantly higher VEGF concentration in serum, compared to matched plasma concentrations, may be due to release of VEGF from platelets during the Previously, most clinical studies have addressed the prognostic impact of preoperative serum VEGF concentrations. Recently, it was demonstrated that plasma VEGF was increased in patients with colorectal cancer compared with controls (


For each increment of 4 metabolic equivalent (MET)-h day−1 of total PA (approximately 1 h daily of moderate effort) cancer incidence (n=3714) tended to be decreased by 2% and cancer mortality decreased significantly by 12% . The 5-year survival after cancer among those men in the top quartile of total PA (77%) was significantly higher compared to the lowest quartile (69%). Compared to those men who hardly ever walked or biked, walking or bicycling an average of 30 min day−1 was associated with a 34% (18–47%) lower rate of cancer death and with improved cancer survival by 33% (14–47%). Incidence of cancer was 16% (2–28%) lower among those who walked or biked at least 60 min day−1. Our results suggest that higher levels of PA and the main component of active living, walking or bicycling are associated with reduced cancer incidence and mortality, as well as higher cancer survival.Within a population-based cohort study, 40 708 men aged 45–79 years followed from 1998 to 2004. After adjusting for potential confounders, we observed a strong inverse linear association between total daily physical activity (PA) and death from cancer ( Cohort studies have suggested that more physically active men, especially those involved in recreational and leisure-time activity, have lower cancer risk and mortality .−1 of moderate activity on most days) showed a significant 17% reduction in cancer mortality compared to inactive participants   aged 45–79 years residing in Västmanland and Örebro counties  received an invitation to participate in the study. An accompanying questionnaire covered PA, current weight, height, education, cigarette smoking, alcoholic beverages, diabetes, family history of cancer, and other lifestyle factors. A total of 48 645 men returned the questionnaire. For the analysis, we excluded those who returned a blank questionnaire (n=92), who died before 1 January 1998 (n=55), and those who had cancer previously  (n=2592). We also excluded heavy manual workers (n=5198) because overall mortality from cancer has been found to be significantly higher among men with manual occupations , home/household work (less than 1 h to more than 8 h day−1), inactive leisure-time watching TV/reading (from 2 h day−1 or less to 5 h day−1 or more), and active leisure time exercising (from less than 1 h to more than 5 h week−1). There was also an open question about the number of sleeping hours per day. To calculate the activity score of specific type of activity, its intensity defined as metabolic equivalents  was multiplied by reported duration (hours)  about duration and intensity of usual PA in the past year. In the questionnaire there were six predefined PA levels for occupational activity  and five to six predefined categories for time spent on different activities: walking/bicycling  and 95% confidence intervals (CIs) for total PA (continuous). We treated total PA both as quartiles and as continuous variables, which allows a more flexible and efficient use of available information. In our main analysis the end points were cancer incidence and cancer mortality. Each participant accrued follow-up time from 1 January 1998 until the date of cancer diagnosis (for incidence) or cancer death , death from any cause, or study end on 31 December 2004, whichever came first. Cancer survival was also studied from diagnosis until death or study end, whichever occurred first.Baseline age-standardised characteristics were presented by quartiles of total PA in MET-h day−2, as continuous) and other potential confounders, including smoking status and pack-years of smoking , alcohol , educational level , diabetes , and parental history of cancer .We adjusted for age (continuous), body mass index  and cancer incidence and mortality rates. Three knot positions were specified for total PA in MET-h dayA total of 75% of participants had complete data on specific types of activity (five questions) included in the total PA score, 13% had one missing value (one of five questions not answered), 3% had two missing values, 3% had three to four missing values, and 6% had five missing values; most of the missing answers regarded occupational activity because many participants was retired. The proportion of missing covariate data was 5% for BMI, and less than 5% for smoking, alcohol, educational level, history of diabetes, and parental history of cancer.−2), and smoking status  and tested the statistical significance of the interactions with the Wald test.A complete analysis was based on 28 880 men in the analytic cohort, and 727 cancer deaths. To evaluate a potential effect of missing values, we used multivariate imputation by chained equations (MICE) to obtain five imputed data sets of the analytic cohort . The RRsvs time since cancer diagnosis. All reported P-values are two-sided; P-values of less than 0.05 were considered statistically significant. All statistical analyses were performed with Stata, version 9.2 .In our secondary analysis among 2551 men with cancer and complete information about PA and confounders, 598 men died from cancer. Cumulative survivor functions for low (bottom), medium (second and third), and high (top quartile) total PA level were estimated using a multivariable Cox regression model and plotted −1), those in the highest quartile were more likely to be never alcohol drinkers and never smokers, and were less likely to have post-secondary education and history of diabetes.Over an average follow-up of 7 years, we identified a total of 3714 incident cancers (263 533 person-years) and 1153 cancer deaths (272 425 person-years). Baseline characteristics of the study population by quartiles of total PA are shown in −1 increment of total PA, which is approximately equivalent to 1 h of moderate effort, was 0.96 (95% CI=0.93–1.00). Further adjustment for BMI, smoking and pack-years, alcohol, education, diabetes, and parental history of cancer slightly attenuated the estimate; each 4 MET-h day−1 increment of PA was associated with an incidence RR of 0.98 (95% CI=0.94–1.01). The test for interaction between smoking status and total PA in predicting cancer was not significant (P=0.83).P=0.41). The multivariate-adjusted MRR in the top quartile (>43 MET-h day−1) of total PA was 0.71 (95% CI=0.58–0.88) as compared to the lowest quartile (<38 MET-h day−1). The highest level of total PA (54 MET-h day−1) was associated with 54% (95% CI=30–70%) lower death rate from cancer compared to those with the lowest level (30 MET-h day−1).Total PA was statistically significantly inversely associated with cancer mortality RRs (MRRs) . The spl−1) based on complete case and multiple imputation analyses are shown in Age and multivariate-adjusted cancer MRRs corresponding to defined increments of total PA (4 MET-h dayP interaction=0.48) as shown graphically in vs current and former smokers.To assess whether the observed inverse relationship with PA reflected reverse causation due to smoking, we stratified the analysis by smoking status , but no −1 increase of total PA were 0.90 (95% CI=0.83–0.97). We also cross-classified participants by age (⩽65 vs >65 years) and BMI (<25 vs 25 kg/m2), but no significant interaction with age (P=0.51), or BMI (P=0.09) emerged.Both the magnitude and direction of the MRRs based on complete information and multiple imputation analyses were overall very similar ; the ave−1, which was significantly higher than 70% for medium  activity levels and 69% for low  activity levels. There was no evidence that these relationships varied for smoking status (P interaction=0.95) as shown in P interaction=0.34).Overall, men in the top quartile of total PA had higher cancer survival  than those in the lowest quartile throughout follow-up, being 77% for men in the top quartile, >43 MET-h day−1 was associated with a nonsignificant trend for a 5% reduction in cancer incidence. An increased duration of walking or bicycling, ranging from 60 to 90 min day−1, was associated with a significant 16% reduction (95% CI=2–28%) in cancer incidence  reduction in cancer mortality in the analytic cohort and a statistically significant 33% (95% CI=14–47%) improvement in cancer survival.Compared to men who hardly ever walked or biked, walking or bicycling an average of 30 min dayncidence . Compare−1, was associated with a significant 34% lower mortality compared to walking or biking hardly ever. Furthermore, cancer survival was improved by 33% among those who walk or biked 30 min day−1. Association of PA with cancer incidence was weak, though, walking or bicycling at least 60 min day−1 was associated with a significant 16% reduction in cancer incidence.In this large cohort study of men, we observed a statistically significant strong inverse dose-response association between total daily PA and cancer mortality. Total daily PA corresponding to 1 h of moderate effort was associated with 12% decrease in cancer mortality. Overall, men in the top PA quartile had higher survival than to those in the lowest quartile, 5-year cancer survival being 77% for high levels at PA and 69% for low levels. The main component of active living, walking or bicycling 30 min dayP for trend=0.02) inverse relationship between cancer mortality and the number of hours per week of activity of at least moderate intensity  aged 50–71 years, which observed a significant  observed a significant protective effect (20%) of combined leisure-time and occupational PA on cancer mortality comparing the highest −1 and cancer incidence, though with longer duration, of at least 60 min was significantly associated with 16% lower risk of cancer. This finding supports the PA recommendation of the World Cancer Research Fund/American Institute for Cancer Research which calls for moderate activity  of 60 min day−1 or more , we found no evidence of effect modification by smoking status. The beneficial effects on cancer survival may reflect reverse causation, if low PA resulted from an under diagnosed cancer with a relatively short survival. If this were true, an interaction between PA and time since diagnosis might be apparent, but no evidence of this was found. Our findings, which may have major public health implications in the prevention and treatment of malignancies, require confirmation.


P = 0.026). No effect of sibship size or birth interval was seen. With one exception (ear infections), these data do not support the hypothesis that a decrease in the occurrence of common childhood infection increases risk of ALL. © 2000 Cancer Research CampaignTo investigate if decreased exposure to common childhood infections is associated with risk of childhood acute lymphoblastic leukaemia (ALL) we conducted a case–control study of 1842 newly diagnosed and immunophenotypically defined cases of ALL under age 15, and 1986 matched controls in the US. Data regarding day care, sibship size and common childhood infections were obtained through parental interviews. Data were analysed stratified by leukaemia lineage and separately for ‘common’ childhood ALL . Neither attendance at day care nor time at day care was associated with risk of ALL overall or ‘common’ ALL. Ear infections during infancy were less common among cases, with odds ratios of 0.86, 0.83, 0.71 and 0.69 for 1, 2–4, 5+ episodes, and continuous infections respectively (trend