Female breast cancer is one of the leading causes of female mortality worldwide. In Malaysia, breast cancer is the most commonly diagnosed cancer in women. Of the women in Malaysia, the Chinese have the highest number of breast cancer cases, followed by the Indian and the Malay. The most common type of breast cancer is infiltrating ductal carcinoma (IDC). A proteomic approach was applied in this study to identify changes in the protein profile of cancerous tissues compared with normal tissues from 18 patients; 8 Chinese, 6 Malay and 4 Indian were analysed. Twenty-four differentially expressed hydrophilic proteins were identified. We evaluated the potential of these proteins as biomarkers for infiltrating ductal carcinoma based on their ethnic-specific expressions. Three of the upregulated proteins, calreticulin, 14-3-3 protein zeta and 14-3-3 protein eta, were found to be expressed at a significantly higher level in the cancerous breast tissues when compared with the normal tissues in cases of infiltrating ductal carcinoma. The upregulation in expression was particularly dominant in the Malay cohort.
The objective of the study was to quantify and to profile the amino acids content in urine samples. The amino acids content in urine was determined in 162 individuals (62 young non-vegetarians aged 15-45 years, 24 elderly non-vegetarians aged 46-70 years, 40 young vegetarians and 36 elderly vegetarians) by high performance liquid chromatography (HPLC). The most common amino acids detected in the young and elderly individuals on vegetarian and non-vegetarian diets were phenylalanine, threonine, arginine and asparagine, while leucine, aspartic acid and alanine were not found in any urine samples in both groups. Isoleucine was not detected in the urine of vegetarians. The concentrations of the majority of essential amino acids were between 0.10 - 2.00 mgl24hrs except for histidine which had a range of 4.1 - 5.0 mgl24hrs. The concentrations of non-essential amino acids varied. Proline, glycine and tyrosine concentrations were between 0.10 - 1.00 mg/24hrs, while cysteine, glutamine, glutamic acid and cystine concentrations were between 11.0 - 21.0 mg124hrs. Asparagine and hydroxy-proline had a range of 0.10 - 5.00 mg/24hrs, while serine and arginine ranged between 31.0 - 50.0 mg124hrs. Isoleucine and serine were not detected in elderly vegetarians while histidine, glycine, glutamic acid and hydroxy-proline were not detected in elderly non-vegetarians. Isoleucine, glycine and hydroxy proline were detected in young non-vegetarians but not in young vegetarians. The levels of amino acids showed no significant statistical differences between young vegetarians and non-vegetarians as well as between elderly vegetarians and non-vegetarians. Phenylalanine, threonine and trypthophan were commonly detected in the lacto-ovo and lacto vegetarians, while valine, cysteine, arginine and asparagine were commonly detected in vegans. In conclusion, except for isoleucine, general differences were seen in urinary amino acid excretions between vegetarians and non-vegetarians even though the differences were statistically not significant. Therefore lacto-ovo diets could be nutritionally adequate as the nutrients were substituted by dairy or plant products.
Matched MeSH terms: Chromatography, High Pressure Liquid
This article examines the fat and fibre intakes of Malaysian adults and highlights discrepancies and practical limitations if these intakes are to match the levels for these nutrients advocated in the World Health Organisation (WHO) and American Heart Association (AHA) ‘diet models’. Local data on food consumption showed that the total fat intakes amongst Malaysian adults, contrary to common perception, were not high and the mean values obtained fell within the range of 40-66g or 22-26% kcal. As such, the dietary target of 30% kcal total fat or its intermediate target of 30-35% kcal, advocated by WHO and AHA mainly to address the problem of a high consumption of dietary fats in western populations, should not be adopted indiscriminately by Malaysians. Dietary fatty acid (FA) analysis by high performance liquid chromatography (HPLC) coupled with the use of food composition tables, showed that the typical Malaysian diet prepared with palm olein or palm olein-groundnut oil blends as cooking oil contained 3.2-4.0% kcal polyunsaturated fatty acids (PUFA), mainly as the w-6 linoleic acid, which is also the predominant essential fatty acid (EFA) in humans. This level of linoleic acid, with an ω-6/ ω--3 FA ratio approximating 10, is adequate for basal PUPA and EFA needs but fell short of the 4-10% kcal linoleic acid recommended by WHO (1993) to counter the effects of the cholesterol-raising saturated fatty acids (SFA). This raised upper limit of 10% kcal linoleic acid (previously 7% kcal), which equals the level of PUFA implied in the AHA diet model, appears unnecessarily high considering that the cholesterol-lowering potential of linoleic acid is maximum at about 6% kcal, while the health hazards associated with long-term high intakes of PUPA have never been completely dismissed. The new WHO lower limit for dietary linoleic acid (4% kcal) would have a controversial impact of raising the previous minimal 3% kcal EFA to above 4% kcal (linoleic + alpha-linolenic acids). Similarly, the WHO recommendation for total dietary fibre of 27-40g (equivalent to a daily combined intake of 400g of vegetables and fruits, 30g of which should come from pulses) appears at present, too high a dietary target for the average Malaysian adult whose habitual daily diet was estimated to contain about 180g of vegetables plus fruits, providing only about 13-16g total dietary fibre. Appropriately, an expert panel on Malaysian Dietary Guidelines has recommended instead, 20-30% kcal total fat containing 3-7% kcal PUFA, and 20-30g total dietary fibre for the local population.
Matched MeSH terms: Chromatography, High Pressure Liquid
Research on natural products has been widely used as a strategy to discover new drugs with potential for applications in complementary medicines because they have fewer side effects than conventional drugs. The aim of the present study was to evaluate the in vitro cytotoxic effects of crude aqueous Catharanthus roseus extract on Jurkat cells and normal peripheral blood mononuclear cells (PBMCs). The aqueous extract was
standardised to vinblastine by high performance liquid chromatography (HPLC) and was used to determine cytotoxicity by the MTS [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. DNA fragmentation assay was employed to determine if cell death was due to apoptosis. The results showed that the aqueous extract induced cell death of Jurkat cells at 24, 48 and 72 hours posttreatment in a time- and dose-dependent manner. However, cells treated at 48 and 72 hours produced higher cytotoxic effects with half maximal inhibitory concentration (IC50)values of 2.55 µg/ml and 2.38 µg/ml, respectively. In contrast, the extract induced normal PBMC proliferation, especially after 24 hours treatment with 1000 µg/ml. This result indicates that the C. roseus crude aqueous extract showed differential effects of inhibiting the proliferation of the Jurkat cell line and promoting the growth of PBMCs. These data suggest that the extract may be applicable for modulating the normal and transformed immune cells in leukaemia patients.
Matched MeSH terms: Chromatography, High Pressure Liquid
Mud lobsters are crustaceans from the genus Thalassina which are lesser known and seldom seen but are nevertheless an important organism to the mangrove ecosystem. In Malaysia and Thailand, mud lobsters are eaten by locals as treatment for asthma. It is traditionally believed that they are effective in reducing the number of asthma attacks and severity of asthma symptoms. However, the therapeutic potential of mud lobster extract remains unclear and has not been fully elucidated or reported in any scientific study. The objectives of this study are to investigate the anti-inflammatory potential of mud lobster, Thalassina anomala extracts (hexane, chloroform and methanol) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages, and to identify the potential bioactive compounds involved. An MTT assay was performed to determine the cytotoxicity of the T. anomala extracts on RAW 264.7 macrophages. Nitrite quantification assay and enzyme-linked immunosorbent assay (ELISA) were conducted to investigate the ability of the T. anomala extracts to suppress the secretion and expression of nitric oxide (NO), Prostaglandin E2 (PGE2) and proinflammatory cytokines (TNF-α, IL-6 and IL-1β) in LPS-stimulated macrophages. GC-MS analysis was done to identify putative metabolites. The hexane extract of T. anomala showed anti-inflammatory activity by significantly inhibiting the LPS-induced production of NO, PGE2, interleukin- (IL-) 6, IL-1β and tumour necrosis factor-alpha (TNF-α) in a concentration-dependent manner. Hexane extract treatment with 100 μg/mL has decreased the NO secretion into 37 μM. Meanwhile, hexane extract at concentration of 100 μg/mL able to significantly suppressed PGE2,TNF-α, IL-6 and IL-1β production into 2015 pg/mL, 2406 pg/mL, 460 pg/mL and 9.6 pg/mL, respectively. GC-MS analysis of the hexane extract revealed the presence of 19 putative compounds. The identified compounds were reported to have anti-inflammatory, antioxidant and antibacterial activities. These results suggest that the hexane extract of T. anomala potentially has anti-inflammatory properties and concentration dependently suppressed NO, PGE2 and proinflammatory cytokines' production in LPS-stimulated macrophages. The findings provide a rational basis of the traditional use of mud lobster for inflammation-associated ailments.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Nutritional quality of the hepatopancreas and gonads of orange portunid mud crab, Scylla olivacea was evaluated for each gender under four treatment of different water velocities (0, 20, 40 and 60 cm s-1), in terms of nutrient reserve and nutrient for reproduction. About 56 crabs were used in this study in which fatty acids composition was analysed using gas chromatography mass-spectrometry (GC-MS). For hepatopancreas analysis, monounsaturated fatty acids (MUFAs) were present in the highest fatty acids concentration, followed by polyunsaturated fatty acids (PUFAs) and, saturated fatty acids (SFAs). However, long-chain polyunsaturated fatty acids (LC-PUFAs) were displayed in low concentration in the hepatopancreas. Total fatty acid (TFAs) composition was significantly higher at moderate velocity of 20 cm s-1 compared to other water velocity treatments. For gonad analysis, 20 cm s-1 showed the highest TFA concentration of 93.34 mg g-1 while, the lowest concentration of 3.90 mg g-1 occurred at 0 cm s-1. There were significant differences in male and female crab's fatty acids contents of gonads at all flow velocities challenged (p < 0.05). PUFAs and MUFAs were dominant while, SFAs were observed at low concentration. This study revealed that, concentration of PUFAs increased as gonad maturation increased. The decreasing concentration of hepatopancreas fatty acids over the culture period indicated that nutrient was shifted from the hepatopancreas, to be used as energy reserved to gonads for further growth of eggs and offspring. The linkages between water flow strength, hepatopancreas, and gonad fatty acids concentrations, is fundamental knowledge useful in establishing efficient habitat velocities selection which will improve aquaculture production of mud crabs with high quality broodstock.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
Mycotoxins are naturally occurring food toxins worldwide that can cause serious health effects. The measurement of mycotoxin biomarkers in biological fluids is needed to assess individuals' exposure. The aim of this study was to investigate the incidence of mycotoxins in the Qatari population. Serum samples from 412 adults and urinary samples from 559 adults were analyzed for the presence of mycotoxin biomarkers. Multimycotoxin approaches have been applied, using liquid chromatography mass spectrometry methods. Samples were further analyzed for the oxidative stress markers and compared with regard to the incidence of mycotoxins. The presence of mycotoxins was identified in 37% of serum samples and in less than 20% of urine samples. It was found that 88% of positive of the samples were positive for only one mycotoxin, while 12% of positive samples had two or more mycotoxins. Trichothecenes and zearalenone metabolites were most commonly detected mycotoxins, followed by aflatoxins, roquefortine C and mycophenolic acid. The presence of mycotoxins was found to positively correlate with oxidative stress markers. The obtained results illustrate the importance of mycotoxin biomonitoring studies in humans and the need to elucidate the underlying mechanisms of mycotoxin-induced toxicity.
Matched MeSH terms: Chromatography, High Pressure Liquid
The study aimed to establish the detection method for bound 3-, 2-MCPD, and glycidol using accelerated solvent extraction (ASE) and gas chromatography mass spectrometry (GC-MS). The ASE was modified for reduced solvent volume and process time to extract lipid from the chocolate spread, infant formula, potato chips, and sweetened creamer. The solvent selected for ASE was a mixture of iso-hexane and acetone at 100°C with the lipid and analyte recovery ranging from 96.9% to 98.6% and 84.1% to 107.5%, respectively. The derivatisation of analytes was adopted from the AOCS method Cd29a-13 for GC-MS analysis. The results showed that the coefficient of determination (R2) of all analytes was >0.99. The limit of detection (LOD) was 0.1 mg kg-1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.2 mg kg-1 expressed in lipid basis for bound glycidol. The limit of quantitation (LOQ) was 0.3 mg kg-1 expressed in lipid basis for both bound 3- and 2-MCPD and 0.6 mg kg-1 expressed in lipid basis for bound glycidol. A blank spiked with 3-monochloropropanediols fatty acid esters (MCPDE) and 2-MCPDE (0.3, 2.1, and 7.2 mg kg-1) and glycidol esters (0.6, 4.7, and 16.6 mg kg-1) were chosen for accuracy and precision tests. The recoveries were 91.7% to 105.9%. Both repeatability and within-laboratory reproducibility of the analysis were within the acceptable level of precision ranging from 1.7% to 16%. This is the first time that a full validation procedure extending to both accuracy and precision tests has been carried out for sweetened creamer and chocolate spread. Overall, the combined protocol of ASE and AOCS Cd29a-13 was successfully validated for both solid and liquid food samples with lipid content from 10% to 30%.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
The chemical composition of the essential oil of Knema kunstleri Warb. (Myristicaceae) was investigated for the first time. The essential oil was obtained by hydrodistillation and fully characterized by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS). In total, 36 components were identified in the essential oil, which made up 91.7% of the total oil. The essential oil is composed mainly of β-caryophyllene (23.2%), bicyclogermacrene (9.6%), δ-cadinene (7.3%), α-humulene (5.7%), and germacrene D (4.3%). The essential oil showed moderate activity towards DPPH free-radical scavenging and lipoxygenase inhibition. To the best of our knowledge, this is the first study of the composition and bioactivities of the essential oil report concerning the genus Knema.
Matched MeSH terms: Gas Chromatography-Mass Spectrometry
The major hemorrhagin (termed hannahtoxin) of the venom of Ophiophagus hannah (king cobra) was purified to electrophoretic homogeneity by DEAE-Sephacel ion exchange chromatography, Sephadex G-200 gel filtration followed by a second DEAE-Sephacel chromatography. Proteolytic activity was associated with the hemorrhagic activity throughout the purification procedures. Hannahtoxin constituted approximately 2% of the crude venom. It had an isoelectric point of 5.3, a carbohydrate content of 12%, a mol. wt of 66,000 as determined by SDS-polyacrylamide gel electrophoresis and 63,000 as determined by gel filtration. It contains 1 mole of Zn per mole of protein. The minimum hemorrhage doses for hannahtoxin are 0.7 microgram and 75 micrograms, respectively, in rabbits and in mice. Hannahtoxin was not lethal to mice at a dose of 2 mg/kg (i.v.) but killed rabbits at doses above 0.18 mg/kg (i.v.). It liberated protein from rabbit glomerular basement membrane but not rat glomerular basement membrane. Treatment of the hemorrhagin with EDTA and 1,10-phenanthroline eliminated both the proteolytic and hemorrhagic activities completely.
The hump-nosed pit viper, Hypanle hypnale, contributes to snakebite mortality and morbidity in Sri Lanka. Studies showed that the venom is hemotoxic and nephrotoxic, with some biochemical and antigenic properties similar to the venom of Calloselasma rhodostoma (Malayan pit viper). To further characterize the complexity composition of the venom, we investigated the proteome of a pooled venom sample from >10 Sri Lankan H. hypnale with reverse-phase high performance liquid chromatography (rp-HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and peptide sequencing (tandem mass-spectrometry and/or N-terminal sequencing). The findings ascertained that two phospholipase A2 subtypes (E6-PLA2, W6-PLA2) dominate the toxin composition by 40.1%, followed by snake venom metalloproteases (36.9%), l-amino acid oxidase (11.9%), C-type lectins (5.5%), serine proteases (3.3%) and others (2.3%). The presence of the major toxins correlates with the venom's major pathogenic effects, indicating these to be the principal target toxins for antivenom neutralization. This study supports the previous finding of PLA2 dominance in the venom but diverges from the view that H. hypnale venom has low expression of large enzymatic toxins. The knowledge of the composition and abundance of toxins is essential to elucidate the pathophysiology of H. hypnale envenomation and to optimize antivenom formulation in the future.
Matched MeSH terms: Chromatography, High Pressure Liquid
The specificity and sensitivity of an indirect and two (an 'ordinary' and a 'rapid') double sandwich enzyme-linked immunosorbent assay (ELISA) procedures for the quantitation of Calloselasma rhodostoma (Malayan pit viper) venom were examined. The three assays were equally sensitive and the accuracy of the assays was not substantially affected by individual variation in the venom composition. The specificity of the assays was examined against 26 venoms from snakes of the families Viperidae and Elapidae. While the double sandwich ELISA procedures were sufficiently specific to be used in the clinical immunodiagnosis of C. rhodostoma bite in Malaysia, the indirect ELISA procedure exhibited extensive cross-reactivity with other Malaysian pit viper venoms. Attempts were made to improve the specificity of the indirect ELISA procedure for the quantitation of C. rhodostoma venom. A 'low ELISA cross-reactivity' venom fraction (termed VF52) was isolated from C. rhodostoma venom by repeated Sephadex G-100 gel filtration chromatography. The indirect ELISA procedure using antibodies to VF52 as immunoreagent showed an improvement in specificity. The use of the indirect ELISA procedure for the detection of C. rhodostoma antibodies was also examined and the results show that the assay was sufficiently specific to be used for retrospective diagnosis of C. rhodostoma bite in Malaysia, in particular when VF52 was used as the coating antigen.
Four tropical PSP toxins-producing dinoflagellates, Alexandrium minutum, Alexandrium tamiyavanichii, Alexandrium tamarense and Alexandrium peruvianum from Malaysian waters were studied to investigate the influences of salinity on growth and toxin production. Experiments were conducted on constant temperature 25 degrees C, 140 microE mol m(-2) s(-1) and under 14:10 light:dark photo-cycle with salinity ranged from 2 to 30 psu. The PSP-toxin congeners, GTX 1-6, STX, dcSTX, NEO and C1-C2 were analysed by high performance liquid chromatography. Salinity tolerance of the four species in decreasing order is A. minutum>A. peruvianum>A. tamarense>A. tamiyavanichii. Specific growth rates and maximum densities varied among these species with A. minutum recorded as the highest, 0.5 day(-1) and 6 x 10(4) cells L(-1). Toxin content decreased with elevated salinities in A. minutum, the highest toxin content was about 12 fmole cell(-1) at 5 psu. In A. tamiyavanichii, toxin content peaked at optimal growth salinity (20 and 25 psu). Toxin content of A. tamarense, somehow peaked at sub-optimal growth salinity (15 and 30 psu). Results of this study implied that salinity fluctuation not only influenced the growth physiology but also toxin production of these species.
Matched MeSH terms: Chromatography, High Pressure Liquid
Screening of the occurrence of potentially toxic diatoms was carried out at two sites of cage cultures in Tebrau Straits, Johor. Phytoplankton samples from Sungai Pendas and Teluk Sengat were collected using a 20 μm mesh plankton net and salinity was recorded in-situ. Nitzschia and Pseudo-nitzschia cells were isolated and established into clonal cultures. All cultures were tested for domoic acid using HPLC-UV analysis and verified by LC-MS analysis. Three Nitzschia spp. and one Pseudo-nitzschia sp. were identified from these locations. Toxic and non-toxic strains of Nitzschia navis-varingica are found at the cage culture areas. Cellular toxin content in the toxic strain of N. navis-varingica is 1.8 pg cell(-1). This is a new record from Malaysia and this species was isolated from estuarine water with salinity 28 PSU. The discovery of toxic Nitzschia species in Tebrau Straits indicates the potential for domoic acid accumulation in seafood.
Matched MeSH terms: Chromatography, High Pressure Liquid
Artemisia annua L., a medicinal herb, produces secondary metabolites with antimicrobial property. In Malaysia due to the tropical hot climate, A. annua could not be planted for production of artemisinin, the main bioactive compound. In this study, the leaves of three in vitro A. annua L. clones were, extracted and two bioactive compounds, artemisinin and a precursor, were isolated by thin layer chromatography. These compounds were found to be effective in inhibiting the growth of Gram-positive and Gram-negative bacteria but not Candida albicans. Their antimicrobial activity was similar to that of antibactericidal antibiotic streptomycin. They were found to inhibit the growth of the tested microbes at the minimum inhibition concentration of 0.09 mg/mL, and toxicity test using brine shrimp showed that even the low concentration of 0.09 mg/mL was very lethal towards the brine shrimps with 100% mortality rate. This study hence indicated that in vitro cultured plantlets of A. annua can be used as the alternative method for production of artemisinin and its precursor with antimicrobial activities.
The study was aimed to investigate the expression of cytosolic and thiolated proteins of Musca domestica larvae under oxidative stress. Proteins from acute treatment of hydrogen peroxide (LC50 = 21.52% (v/v)) on 3rd stage larvae of housefly were extracted and purified using an activated Thiol Sepharose® for thiolated protein purification. Two dimensional gel electrophoresis was used for visualizing and analyzing expression of cytosolic and thiolated proteins. Protein spots with more than 5 fold of expression change were identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). The cytosolic proteins were actin, tropomyosin, ubiquitin, arginine kinase, pheromone binding protein/general odorant binding protein, and ATP: guanidino phosphotransferase. The thiolated proteins with more than 5 fold change in expression as an effect to the acute treatment were fructose bisphosphate aldolase, short chain dehydrogenase and lactate/malate dehydrogenase. The proteins identified in the study should provide vital information for future reference in oxidative stress defence and response occurring in houseflies.
A selective and sensitive HPLC assay for the quantitative determination of a new antifilarial drug, 6,4'-bis-(2-imidazolinylhydrazone)-2-phenylimidazo[1,2-a]pyr idine (CDR 101) is described. After extraction from plasma and blood, CDR 101 was analysed using a C18 Nucleosil ODS column (250x4.6 mm, 5 microm particle size) and mobile phase of acetonitrile-0.05 M ammonium acetate adjusted to pH 3.0, with UV detection at 318 nm. The mean recoveries of CDR 101 in plasma and blood over a concentration range of 25-500 ng/ml were 95.5+/-2.01% and 83.3+/-1.87%, respectively. The within-day and day-to-day coefficient of variations for plasma were 3.23-6.21% and 2.59-9.90%, respectively, those for blood were 2.59-5.92% and 2.89-6.82%, respectively. The minimum detectable concentration for CDR 101 was 1 ng/ml in plasma and 2.5 ng/ml in whole blood. This method was found to be suitable for clinical pharmacokinetic studies.
Matched MeSH terms: Chromatography, High Pressure Liquid
A simple high-performance liquid chromatographic method using fluorescence detection was developed for the determination of acyclovir in human plasma. The method entailed direct injection of the plasma sample after deproteination. It is both specific and sensitive with a detection limit of 30 ng/ml at a signal-to-noise ratio of 3:1, and is thus suitable for use in pharmacokinetic studies of acyclovir. The method had a mean absolute recovery of 96%, while the within-day and between-day coefficients of variation and percentages error were all less than 8%. The calibration curve was linear over a concentration range of 62.5-4000 ng/ml.
Matched MeSH terms: Chromatography, High Pressure Liquid