Amitha K. Hewavitharana

Discovery of a New Source of Rifamycin Antibiotics in Marine Sponge Actinobacteria by Phylogenetic Prediction

ABSTRACT Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria.

Matrix matching in liquid chromatography-mass spectrometry with stable isotope labelled internal standards--is it necessary?

Matrix matching is used in analysis to compensate for matrix effects that influence analytical response. It has been a widely discussed topic in electro-spray mass spectrometry where the ionization suppression is a major problem in accurate quantitative analysis. However, the unique strength of mass spectrometry to detect and quantify accurately a co-eluting stable isotope labelled internal standard offers an easy solution to the ionization suppression problem. Given the fact that it is impossible to match the matrix of the calibration standards with all samples, mass spectrometry allows accurate quantitation without the need for matrix matching, as long as the internal standard co-elutes with the analyte of interest. If the analyte and internal standard co-elute, the slope of the calibration curve analyte response/internal standard vs. analyte concentration is independent of the matrix composition, eliminating the need for matrix matching.

Monitoring potential prostate cancer biomarkers in urine by capillary electrophoresis–tandem mass spectrometry

Current prostate cancer (PCa) diagnosis based on prostate-specific antigen (PSA) has been gradually losing its credibility over the last decade due to contradictory results in published literature and clinical practice. Recently, a group of potential PCa biomarkers in urine, particularly sarcosine, was found to increase significantly as the cancer progressed to metastasis. We report a simple, robust, and reproducible CE-ESI-MS/MS method for the determination of sarcosine and other representative potential biomarkers in pooled urine. The pooled urine was obtained from 20 healthy adult volunteers between the ages of 23-30 years old. A solid phase extraction (SPE) technique was optimized for maximum recovery of sarcosine. With no derivatization step, excellent resolution between sarcosine and its isomers (α-alanine and β-alanine) was achieved. A separate non-SPE method was also developed for quantitative determination of highly concentrated urinary metabolites. CE separation was performed on a positively-charged, polyethyleneimine (PEI)-coated capillary using 0.4-2% formic acid in 50% methanol. Precision for intra- and inter-day standard addition calibration of sarcosine were found to be within 15%, whereas intra-day precisions for the rest of the metabolites varied from 0.03 to 13.4%. Acceptable intra-day and inter-day accuracies, ranging from 80 to 124%, were obtained for sarcosine and the other metabolites.

Disruption of NaS1 sulfate transport function in mice leads to enhanced acetaminophen-induced hepatotoxicity.

Sulfate is required for detoxification of xenobiotics such as acetaminophen (APAP), a leading cause of liver failure in humans. The NaS1 sulfate transporter maintains blood sulfate levels sufficiently high for sulfonation reactions to work effectively for drug detoxification. In the present study, we identified two loss‐of‐function polymorphisms in the human NaS1 gene and showed the Nas1‐null mouse to be hypersensitive to APAP hepatotoxicity. APAP treatment led to increased liver damage and decreased hepatic glutathione levels in the hyposulfatemic Nas1‐null mice compared with that in normosulfatemic wild‐type mice. Analysis of urinary APAP metabolites revealed a significantly lower ratio of APAP‐sulfate to APAP‐glucuronide in the Nas1‐null mice. These results suggest hyposulfatemia increases sensitivity to APAP‐induced hepatotoxicity by decreasing the sulfonation capacity to metabolize APAP. In conclusion, the results of this study highlight the importance of plasma sulfate level as a key modulator of acetaminophen metabolism and suggest that individuals with reduced NaS1 sulfate transporter function would be more sensitive to hepatotoxic agents. (HEPATOLOGY 2006;43: 1241–1247.)

Mango extracts and the mango component mangiferin promote endothelial cell migration.

This study tested the hypothesis that mango extracts contain bioactive molecules capable of modulating endothelial cell migration, an essential step in the formation of new blood vessels or angiogenesis. The formation of new blood vessels is an important therapeutic target for diseases such as limb ischemia, coronary infarction or stroke. We examined the effect of mango peel and flesh extracts as well as the individual polyphenolic molecules, mangiferin and quercetin, on bovine aortic cell migration using a modified Boyden chamber assay. Our results show that mangiferin, and extracts rich in mangiferin, increase endothelial cell migration. The dose-effect relationship for various extracts further suggests that this action of mangiferin is modulated by other components present in the extracts. The promigratory effect of mango extracts or mangiferin was unrelated to an effect on cell proliferation, and did not involve a change in the production of matrix metalloprotease-2 or -9 by the endothelial cells. Taken together, these results suggest that mangiferin present in mango extracts may have health promoting effects in diseases related to the impaired formation of new blood vessels.

Simultaneous liquid chromatographic determination of vitamins A, E and β-carotene in common dairy foods

Milkfat was extracted quantitatively from dairy foods and the vitamins A, E and beta-carotene in the extract were analysed by normal phase high performance liquid chromatography (HPLC) using a silica column, a fluorescence detector and a UV/visible detector. Products such as butter and anhydrous milkfat were dissolved simply in hexane, whereas milk and milk powders underwent a Rose-Gottlieb fat extraction prior to HPLC analysis. The extraction method used as superior to commonly used saponification procedures in terms of speed as well as reduced loss and isomerization of the vitamin components in the food, because the chemical form of the vitamin is not changed to a less stable form such as retinol during extraction. The individual geometrical isomers of vitamin A, which possess different in vivo vitamin activities, were uniquely identified and quantified. Recovery studies showed satisfactory results for all three vitamins. The simultaneous nature of the assay allowed the estimation of the total vitamin A activity of the food because both vitamin A and pro-vitamin A (beta-carotene) were quantified. The single extraction and single HPLC analysis used for the three vitamins made the method well suited for routine work

Effect of dietary tocopherols and tocotrienols on the antioxidant status and lipid stability of chicken

We determined the effect of dietary tocopherols and tocotrienols on the lipid stability of pre-cooked chicken breast and thigh. The birds were supplemented with one of two doses of a commercial mixture of tocopherols and tocotrienols (Oryza1, Oryza2) or one of two doses of all-rac α-tocopherol acetate (Toc1, Toc2). Diets were formulated so that Oryza1 and Toc1 and Oryza2 and Toc2 contained similar tocopherol concentrations. No quantifiable amounts of tocotrienols were found in either breast or thigh muscles. Tocotrienols present in the diet reduced muscle α-tocopherol concentration. The effect of Oryza1 on the tocopherol content in muscle and on its lipid stability was not significant. The Oryza2, Toc1 and Toc2 diets increased the α- and γ-tocopherol in breast and thigh muscles and enhanced their lipid stability. This improvement was only due to the antioxidant action of the tocopherols. Lipid stability of pre-cooked chicken was not enhanced by adding tocotrienols to a tocopherol supplement.

Re: Florian Jentzmik, Carsten Stephan, Kurt Miller, et al. Sarcosine in Urine After Digital Rectal Examination Fails as a Marker in Prostate Cancer Detection and Identification of Aggressive Tumours. Eur Urol 2010;58:12–8

Background Sarcosine in urine was recently suggested to be a promising tool in prostate cancer (PCa) diagnostics. Objective To reevaluate sarcosine as a potential biomarker for early PCa detection and for prediction of tumour aggressiveness. Design, setting, and participants Sarcosine was measured in urine samples from 106 PCa patients and 33 patients with no evidence of malignancy (NEM), confirmed by 8–12 core prostate biopsies, after standardised digital rectal examination, as well as from 12 healthy men and women. The results were related to the clinicopathologic data on prostate volume, tumour stage, Gleason score, and prostate specific antigen (PSA). Measurements Sarcosine in urine was determined by gas chromatography-mass spectrometry using a commercial amino acid assay and was normalised to urine creatinine. Nonparametric statistical tests and receiver operating characteristics (ROC) analyses were performed to assess the diagnostic performance. Results and limitations The median sarcosine-creatinine ratio in urine was 13% lower in PCa than in NEM patients. Sarcosine values were not associated with tumour stage (pT2 vs pT3) or grade (Gleason score <7 vs ≥7). ROC analyses proved that the discrimination between PCa and NEM patients was not improved by sarcosine in comparison with total PSA, but it was significantly worse than the percent free PSA. The higher proportion of PCa than NEM patients can be considered a limitation of this study. Conclusions Sarcosine in urine after rectal digital examination cannot be considered as a suitable marker to differentiate between patients with and without PCa. Take Home Message The results of this study indicate that, contrary to a recent report, sarcosine in urine after digital rectal examination cannot be considered a suitable marker for prostate cancer detection or for identification of aggressive cancer types.

Discovering the recondite secondary metabolome spectrum of Salinispora species: a study of inter-species diversity.

Patterns of inter-species secondary metabolite production by bacteria can provide valuable information relating to species ecology and evolution. The complex nature of this chemical diversity has previously been probed via directed analyses of a small number of compounds, identified through targeted assays rather than more comprehensive biochemical profiling approaches such as metabolomics. Insights into ecological and evolutionary relationships within bacterial genera can be derived through comparative analysis of broader secondary metabolite patterns, and this can also eventually assist biodiscovery search strategies for new natural products. Here, we investigated the species-level chemical diversity of the two marine actinobacterial species Salinispora arenicola and Salinispora pacifica, isolated from sponges distributed across the Great Barrier Reef (GBR), via their secondary metabolite profiles using LC-MS-based metabolomics. The chemical profiles of these two species were obtained by UHPLC-QToF-MS based metabolic profiling. The resultant data were interrogated using multivariate data analysis methods to compare their (bio)chemical profiles. We found a high level of inter-species diversity in strains from these two bacterial species. We also found rifamycins and saliniketals were produced exclusively by S. arenicola species, as the main secondary metabolites differentiating the two species. Furthermore, the discovery of 57 candidate compounds greatly increases the small number of secondary metabolites previously known to be produced by these species. In addition, we report the production of rifamycin O and W, a key group of ansamycin compounds, in S. arenicola for the first time. Species of the marine actinobacteria harbour a much wider spectrum of secondary metabolites than suspected, and this knowledge may prove a rich field for biodiscovery as well as a database for understanding relationships between speciation, evolution and chemical ecology.

Bioactivity of Mango Flesh and Peel Extracts on Peroxisome Proliferator‐Activated Receptor γ[PPARγ] Activation and MCF‐7 Cell Proliferation: Fraction and Fruit Variability

Mangos are a source of bioactive compounds with potential health promoting activity. Biological activities associated with mango fractions were assessed in cell-based assays to develop effective extraction and fractionation methodologies and to define sources of variability. Two techniques were developed for extraction and fractionation of mango fruit peel and flesh. Liquid chromatography-mass spectrometry (LC-MS) was used to assess compositional differences between mango fractions in flesh extracts. Many of the extracts were effective in inhibiting the proliferation of human breast cancer cells in vitro. All fractions showed bioactivity in PPAR activation assays, but quantitative responses showed marked fruit-to-fruit variability, highlighting the need to bulk fruit prior to extraction for activity-guided fractionation of bioactive components. This study also suggests that combinations of diverse molecular components may be responsible for cell-level bioactivities from mango fractions, and that purification and activity profiling of individual components may be difficult to relate to whole fruit effects. Practical Application: Although the health benefits of fruits are strongly indicated from studies of diet and disease, it is not known what role individual fruit types can play, particularly for tropical fruits. This study shows that there is a diversity of potentially beneficial bioactivities within the flesh and peel of mango fruit, although fruit-to-fruit variation can be large. The results add to the evidence that the food approach of eating all components of fruits is likely to be more beneficial to health than consuming refined extracts, as the purification process would inevitably remove components with beneficial bioactivities.