Enzymatic Kit Research Articles

Standard Gibbs Energy of Metabolic Reactions: I. Hexokinase Reaction.

The standard Gibbs energy of reaction enables calculation of the driving force of a (bio)chemical reaction. Gibbs energies of reaction are required in thermodynamic approaches to determine fluxes as well as single reaction conversions of metabolic bioreactions. The hexokinase reaction (phosphorylation of glucose) is the entrance step of glycolysis, and thus its standard Gibbs energy of reaction (ΔRg°) is of great impact. ΔRg° is accessible from equilibrium measurements, and the very small concentrations of the reacting agents cause usually high error bars in data reduction steps. Even worse, works from literature do not account for the nonideal behavior of the reacting agents (activity coefficients were assumed to be unity); thus published ΔRg° values are not standard data. Consistent treatment of activity coefficients of reacting agents is crucial for the accurate determination of standard Gibbs energy from equilibrium measurements. In this work, equilibrium molalities of hexokinase reaction were measured with an enzyme kit. These results were combined with reacting agents' activity coefficients obtained with the thermodynamic model ePC-SAFT. Pure-component parameters for adenosine triphosphate (ATP) and adenosine diphosphate (ADP) were fitted to experimental osmotic coefficients (water + Na2ATP, water + NaADP). ΔRg° of the hexokinase reaction at 298.15 K and pH 7 was found to be -17.83 ± 0.52 kJ·mol-1. This value was compared with experimental literature data; very good agreement between the different ΔRg° values was obtained by accounting for pH, pMg, and the activity coefficients of the reacting agents.

Advanced glycation end products increase lipids accumulation in macrophages through upregulation of receptor of advanced glycation end products: increasing uptake, esterification and decreasing efflux of cholesterol.

BackgroundPrevious reports have suggested that advanced glycation end products (AGEs) participate in the pathogenesis of diabetic macroangiopathy. Our previous study have found that AGEs can increase the lipid droplets accumulation in aortas of diabetic rats, but the current understanding of the mechanisms remains incomplete by which AGEs affect lipids accumulation in macrophages and accelerate atherosclerosis. In this study, we investigated the role of AGEs on lipids accumulation in macrophages and the possible molecular mechanisms including cholesterol influx, esterification and efflux of macrophages.MethodsTHP-1 cells were incubated with PMA to differentiate to be macrophages which were treated with AGEs in the concentration of 300 μg/ml and 600 μg/ml with or without anti-RAGE (receptor for AGEs) antibody and then stimulated by oxidized-LDL (oxLDL) or Dil-oxLDL. Lipids accumulation was examined by oil red staining. The cholesterol uptake, esterification and efflux were detected respectively by fluorescence microscope, enzymatic assay kit and fluorescence microplate. Quantitative RT-PCR and Western blot were used to measure expression of the moleculars involved in cholesterol uptake, synthesis/esterification and efflux.ResultsAGEs increased lipids accumulation in macrophages in a concentration-dependent manner. 600 μg/ml AGEs obviously upregulated oxLDL uptake, increased levels of cholesterol ester in macrophages, and decreased the HDL-mediated cholesterol efflux by regulating the main molecular expression including CD36, Scavenger receptors (SR) A2, HMG-CoA reductase (HMGCR), ACAT1 and ATP-binding cassette transporter G1 (ABCG1). The changes above were inversed when the cells were pretreated with anti-RAGE antibody.ConclusionsThe current study suggest that AGEs can increase lipids accumulation in macrophages by regulating cholesterol uptake, esterification and efflux mainly through binding with RAGE, which provide a deep understanding of mechanisms how AGEs accelerating diabetic atherogenesis.

INFLUENCE OF SERTRALINE ON HYPOGLYCEMIC EFFECT OF ORALLY ADMINISTERED PIOGLITAZONE AND ROSIGLITAZONE IN HEALTHY RABBITS

The present study was planned to study the influence of sertraline on hypoglycemic effect of orally administered pioglitazone and rosiglitazone in healthy rabbits. In the beginning of experiment, the rabbit were fasted for 18 hours with distilled water ad libitum. The blood was withdrawn from the marginal ear vein of pioglitazone (10 mg/kg, p.o.) treated rabbits and rosiglitazone (720 μg/kg, p.o.) treated rabbits at different time intervals at 0 h, 1, 2, 4, 8, 12, 18, 24 h before and after administration of sertraline (18 mg/ kg, p.o). The blood glucose was estimated by GOD-POD enzymatic kit method using semi-autoanalyzer and data were expressed in mg/dl. The results shown that sertraline increases the onset, peak effect and duration action of pioglitazone and rosiglitazone in healthy rabbits during concomitant administration of these drugs. In conclusion the dose and frequency of administration of sertraline with pioglitazone or rosiglitazone should be adjusted while administrating concurrently.

Development of novel bienzymatic assay for quantification of glucose in human serum

Aim: The main objective of this investigation was to design and to develop a simple, sensitive, and convenient assay for quantification of glucose in human serum sample. Materials and Methods: A novel bienzymatic assay for the quantification of glucose by spectrophotometry has been developed. The method is based on the coupling of 4-aminoantipyrine and iminodibenzyl in the presence of bienzyme peroxidase/glucose oxidase (GOx) to form a blue colored product which is monitored for absorbance at 620 nm at 23°C in 100 mmol/L KH2PO4/NaOH buffer of pH 6.3. Results and Discussion: Under optimized assay conditions, glucose could be quantified in a linear range from 1.15 to 562 μmol/L and 2.31 to 370 μmol/L by kinetic and fixed time method. The limit of detection and limit of quantification were measured. Michaelis–Menten constant for glucose was found to Km = 357 μmol/L and maximum rate of the reaction (Vmax) was 0.117 × 10−6 mol/L/min. The proposed method was applied for the quantification of glucose in human serum samples, which has a good regression coefficient of 0.9992 with the enzymatic kit method. The inter-day precision was 0.53-2.9% (n = 10) and intra-day precision was 1.5-3.03% (n = 10). The accuracy range for glucose was fond to be 90-104%. The glucose recovery ranged from 91.92% to 108.5%. Conclusion: The present assay was rapid, sensitive, and convenient for quantification of glucose in human serum sample with good recovery, high accuracy, and minimum interference.

The flower of Edgeworthia gardneri (wall.) Meisn. suppresses adipogenesis through modulation of the AMPK pathway in 3T3-L1 adipocytes

Ethnopharmacological relevanceThe flower of Edgeworthia gardneri (Wall.) Meisn., locally named “Lvluohua, 绿萝花”, has been widely used as Tibetan folk medicine for the treatment of metabolic diseases for a long time. Aim of this studyTo evaluate the anti-adipogenesis effect of ethyl acetate extract of the flower of E. gardneri (EEG extract) in 3T3-L1 adipocytes. Materials and methodsObesity-related parameters such as lipid accumulation and TG content were determined by Oil red O staining and enzymatic kit, respectively. Western blotting was used to determine the expressions of peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein-α (C/EBPα), phosphorylated adenosine 5′-monophosphate (AMP)-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). Moreover, main constituents of EEG extract were analyzed by high performance liquid chromatography (HPLC). ResultsEEG extract decreased the lipid and triglyceride (TG) accumulations during the differentiation process and down-regulated the adipogenesis-related transcriptional factors PPARγ and C/EBPα. EEG extract treatment increased AMPK and ACC phosphorylation. In addition, pretreatment with AMPK inhibitor, weakened the inhibitory effects of EEG extract on the expressions of PPARγand C/EBPα. HPLC analysis indicated that tiliroside was the main constituent in EEG extract. ConclusionsThese results suggest that EEG extract may exert anti-adipogenic effects through modulation of the AMPK signaling pathway.

Construction of Local Isolates of Cyanobacteria for Ethanol Production.

Cyanobacteria can use solar energy and convert carbon dioxide into biofuel molecules in
 one single biological system. In this research, Synechococcus sp. was isolated from
 Saray Subhan Agha fresh water, a pure culture of Synechococcus was obtained by
 several subculturing on BG11 media. For the production of ethanol by Synechococcus
 sp. pyruvate decarboxylase (PDC) and alcohol dehydrogenseII (ADH II), genes from
 Zymomonas mobilis ATCC (29191), were amplified by PCR and cloned into the
 pSyn_1⁄D-TOPO® Vector. The Synechococcus and Synechococcus elongates
 transformed with constructed vector (pSyn_1⁄D-TOPO®) that harboring the two ethanol
 fermenting genes. The transformation was performed using a double homologous
 recombination system to integrate the PDC and ADHII genes into the local isolates of
 Synechococcus sp. and Synechococcus elongatus chromosome under the control
 cyanobacterial weak constitutive nickel inducible promoter. The recombinant
 Synechococcus cells grow in different concentrations of NiSO4 (1, 2.5, 5, 7.5, 10) μM in
 BG11 media, under different temperature (15, 30, 45) ̊C and different light intensity (10,
 50, 150) μE. The enzymatic ethanol assay kit was used to determine ethanol
 concentration produced by both recombinant Synechococcus sp. and recombinant
 Synechococcus elongatus. Highest ethanol concentration obtained by those cultures
 containing five μl NiSO4, which incubated under continues light of 50μE at 30 ̊C
 (Optimum um condition for ethanol production by recombinant Synechococcus cells).
 The amount of ethanol produced by local isolates of Synechococcus sp. was 0.00103 g/l,
 whereas for Synechococcus elongatus was 0.0138 g/l. The amount of ethanol produced
 by those Synechococcus cultures containing different concentrations of NiSO4 were
 incubated under continuous light of (10 and 150) μE and temperature of (15 and 45) ̊C
 was less than those cultures were incubated under light of 50 μE and temperature of 30 ̊C.

Soluble Sugar and Starch Extraction and Quantification from Maize (Zea mays) Leaves.

An easy-to-perform protocol for isolating and quantifying soluble sugars (sucrose, glucose, and fructose) and starch from maize (Zea mays) leaf tissue is described. The method has been optimized to extract non-structural carbohydrates (NSC) from frozen, finely ground tissue in a methanol:chloroform:water solution. Three rounds of tissue extraction for 30min each at 50°C provide quantitative recovery of soluble sugars. The use of alternative extraction solvents is discussed, as well as the advantages and disadvantages of these solvents. Additionally, we provide two support protocols. The first quantifies the isolated NSC via commercially available enzymatic kits that couple the amount of each specific sugar to the production of NADPH, which is detected using equipment readily available to most laboratories. The second describes the preparation of a purification column to remove strongly charged or hydrophobic molecules from the extracted sugar solution, which is required prior to quantification with high-pressure liquid chromatography or high-performance anion-exchange chromatography. The protocols are robust and easily adapted for use in measuring NSC extracted from other plant species or tissues, making them ideal for new users. © 2016 by John Wiley & Sons, Inc.

<i>Ixeris dentata</i> (Thunb) Nakai Ethylacetate Extract Attenuates Sterol Regulatory Element-Binding Proteins-1c via AMP-Activated Protein Kinase Activation

Purpose: To investigate the molecular mechanisms underlying the role of Ixeris dentataa extract (IDE) in the prevention of high glucose induced lipid accumulation in human HepG-2 hepatocytes.Methods: IDE extract was prepared by maceration in ethyl acetate. Its fractionation was carried out by column chromatography. HepG-2 cells were pretreated with various concentration of IDE (0, 10, 20, 40 and 80 μg/mL) and then treated with serum-free medium with normal glucose (5 mM) for 1 h, followed by exposure to high glucose (30 mM D-glucose) for 24 h. Cell viability and cytotoxicity parameters were measured using lactate dehydrogenase (LDH) and MTT assay while triglyceride and total cholesterol levels were evaluated using respective enzymatic reagent kits. Protein expressional levels were analyzed by Western blotting.Results: IDE did not influence the cell viability (up to 200 μg/mL) and did not show any signs of cytotoxicity (up to 80 μg/mlL. IDE significantly attenuated lipid accumulation in human HepG2 hepatocytes when exposed to high glucose (30 mM D-glucose) in a dose-dependent manner (p < 0.05, 0.01 and 0.001 at 20, 40 and 80 μg/mL concentrations, respectively). Nile red staining showed that 10, 20, 40 and 80 μg/mL concentrations of IDE reduced lipid accumulation by 23.4, 34.8 (p < 0.05), 46.5 (p < 0.01) and 53.2 % (p < 0.001), respectively. The increased levels of triglycerides and total cholesterol were also attenuated by IDE (p < 0.001 at 80 μg/mL). Further, IDE attenuated the expression of fatty acid synthase and sterol regulatory element-binding protein-1. Adenosine monophosphate-activated protein kinase was also activated by IDE treatment when exposed to high glucose level (30 mM Dglucose) in human HepG2 hepatocytes.Conclusion: The findings indicate that IDE exerts hypolipidemic effect by inhibiting lipid biosynthesis, mediated via AMPK signaling. This probably explains the extract’s beneficial effect in various inflammation disorders.Keywords: Ixeris dentata extract, Lipogenesis, AMP-activated protein kinase, Sterol regulatory element-binding protein 1 -1, Anti-obesity, HepG-2 cell

Heme oxygenase-1 (HO-1) protects human lens epithelial cells (SRA01/04) against hydrogen peroxide (H2O2)-induced oxidative stress and apoptosis

ObjectivesThis study aimed to investigate the protective role of heme oxygenase-1 (HO-1) in H2O2-induced oxidative stress and apoptosis in human lens epithelial cells (hLEC; SRA01/04). MethodsSRA01/04 cells were exposed to a hydrogen peroxide (H2O2) concentration gradient and inducers of HO-1 such as cobalt protoporphyrin (CoPP) and zinc protoporphyrin (ZnPP), respectively. In addition, an RNA silencing experiment was conducted to investigate the HO-1 function in this study. A Cell Counting Kit-8 (CCK-8) assay was used to measure cell viability. Western blot and ELISA were used to detect the level of HO-1 expression. In our study, hLECs were exposed to 400 μM hydrogen peroxide (H2O2) for 24 h with or without pretreatment with 10μΜ CoPP or 10μΜ ZnPP, respectively. Double immunofluorescence staining was used for cell identification and the qualitative expression of HO-1. Expression of HO-1 was monitored using Western blot and ELISA. Intracellular reactive oxygen species (ROS) were detected by flow cytometry analyses; commercial enzymatic kits were used to measure the levels of glutathione (GSH), as well as superoxide dismutase (SOD). The proportion of cell apoptosis was quantified by annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. The expression of caspase family (−8, -3) proteins was measured by Western blot analysis. ResultsHO-1 significantly restored the cell viability under H2O2 injury via reducing the generation of ROS and increasing the levels of SOD and GSH activity. Moreover, HO-1 also inhibited H2O2-induced caspase-8 and caspase-3 proteins, thus significantly reducing the apoptosis of SRA01/04. An RNA silencing experiment demonstrated the increased resistance of LECs to oxidative stress specifically for increased levels of HO-1. ConclusionsThese findings suggested that HO-1 protects human lens epithelial cells from H2O2-induced oxidant stress by upregulating antioxidant enzyme activity, reducing ROS generation, and thus inhibiting caspase family-dependent apoptosis.

Xylitol Chewing Gums on the Market: Do They Prevent Caries?

To measure the xylitol content in sugar-free chewing gums available on the market in Gulf Cooperation Council (GCC) countries in the Middle East, in order to identify those products that can provide the recommended daily dose of xylitol for caries prevention (6-7 g). Acid production from chewing gums was also measured in vitro and in vivo. Twenty-one chewing gums containing xylitol were identified and collected from the GCC market (Kuwait, Bahrain, Qatar, Saudi Arabia, UAE and Oman). Xylitol was extracted and its concentration was analysed using a special enzymatic kit. The pH of extracts was measured during 30-min incubation with Streptococcus mutans. Changes in saliva and plaque pH were noted in four subjects after the consumption of highly concentrated xylitol gums. The xylitol content in grams was clearly mentioned only on one product's label. Twelve products stated the percentage of xylitol (3.5% to 35%). The rest did not specify the amount. The mean measured weight of one piece of gum was 1.67 ± 0.38 g. The mean measured xylitol content/piece was 0.33 ± 0.21 g. Xylitol content was < 0.3 g/ piece in 9 products, 0.3-0.5 g in 7 and > 0.5 g in 5 products. None of the highly concentrated xylitol gums showed a pH drop in vitro or in vivo. One chewing gum, containing xylitol and glucose, resulted in a low pH level (< 5.5) when tested in vitro. The majority of xylitol chewing gums sold on the GCC market do not provide the consumers with the recommended daily dose of xylitol for caries prevention. Clear, accurate labeling is recommended.

Involvement of Nuclear Related Factor 2 Signaling Pathway in the Brain of Obese Rats and Obesity-Resistant Rats Induced by High-Fat Diet.

We aimed to investigate the mechanism of brain damage in diet-induced obese (DIO) rats and diet-resistant (DR) rats from the viewpoint of redox state and nuclear related factor 2 (Nrf2) signaling pathway. Sprague-Dawley rats were fed with a high-fat diet for 10 weeks to obtain the DIO and DR rats. d-Galactose was injected subcutaneously through the back of the neck for 10 weeks to establish oxidative stress model rats. Then, the ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) and the level of glutathione peroxidase (GSH-Px) in serum and brain tissue were measured by using enzymatic assay kits. The levels of cholecystokinin and peptide YY in the brain tissue were detected by using enzyme-linked immunosorbent assay kits. In addition, the protein expression of Nrf2 and its downstream factors such as heme oxygenase 1, manganese superoxide dismutase, and NAD(P)H quinone oxidoreductase 1 (NQO1) in the brain tissue were measured by Western blotting. In the brain of DIO rats, the level of GSH and ratio of GSH/GSSG were lower, whereas the GSH-Px concentration was higher compared with DR rats significantly. On the other hand, the GSSG level was higher in the serum of DIO rats compared with the DR rats. The oxidative stress state in the brain of DIO rats, but not in DR rats, were observed. In addition, the protein expressions of Nrf2 and NQO1 were downregulated in the brain of DR rats compared with that in DIO rats. Our data suggest that the Nrf2/NQO1 signaling pathway and redox state were involved in the pathogenesis of the rats prone to obesity, but not the DR rats resistant to obesity.

Characterization of the Key Aroma Compounds in Two Commercial Rums by Means of the Sensomics Approach.

Two rums differing in their overall aroma profile and price level (rum A, high price; rum B, low price) were analyzed by means of the Sensomics approach. Application of aroma extract dilution analysis (AEDA) on a distillate of volatiles prepared from rum A revealed 40 aroma-active compounds in the flavor dilution (FD) factor range from 8 to 2048. The identification experiments indicated cis-whiskey lactone, vanillin, decanoic acid, and 2- and 3-methylbutanol with the highest FD factors. The AEDA of a distillate prepared from rum B showed only 26 aroma-active compounds in the same FD factor range. Among them, in particular, ethyl butanoate, 1,1-diethoxyethane, ethyl (S)-2-methylbutanoate, and decanoic acid appeared with the highest FD factors. Thirty-seven compounds having at least an FD factor ≥32 in one of the two rums were quantitated using stable isotope dilution assays or enzyme kits (2 compounds). The calculation of odor activity values (OAVs; ratio of concentration to respective odor threshold) indicated ethanol, vanillin, ethyl (S)-2-methylbutanoate, and (E)-β-damascenone with the highest OAVs in rum A, whereas ethanol, 2,3-butanedione, 3-methylbutanal, and ethyl butanoate revealed the highest OAVs in rum B. Most compounds were present in similar concentrations in both rums, but significant differences were determined for vanillin, cis-whiskey lactone, and 4-allyl-2-methoxyphenol (all higher in rum A) and 3-methylbutanal, 2,3-butanedione, and ethyl butanoate (all higher in rum B). Finally, the aromas of both rums were successfully simulated by a recombinate using reference odorants in the same concentrations as they naturally occurred in the spirits.

Standardization of Enzymatic Diagnostic Kit based method for cholesterol estimation in Butter, Kalakand, Khoa and Paneer

The objective of this study were to standardize the protocol for cholesterol quantification in dairy based products (butter, kalakand, khoa and paneer) using enzymatic diagnostic kit (EDK) assay. Practically about 0.15-0.2 g product was saponified using different methanolic KOH (2.5%, 5% and 7.5%) as well as at different saponification temperature (80 0 C, 85 0 C and 90 0 C) for 20 min. followed by extraction of unsaponifiable in hexane. An aliquot of unsaponifiable fraction was evaporated at 50 0 -65 0 C and cholesterol estimation was done using EDK. For standardization of protocol recovery trials also conducted in dairy based products at different methanolic KOH as well as at different saponification temperature. The optimized conditions for rapid saponification of Dairy products were found to be the use of 5% methanolic KOH and heating to 90 0 C for 20 min., wherein the per cent recovery in case of butter, kalakand, khoa and paneer were to the tune of 100.13±0.70; 100.25±0.58; 100.54±0.51; 97.99±0.28, respectively. Recovery studies clearly indicated that developed EDK based method is a choice for the rapid determination of cholesterol content in selected indigenous dairy products. On using the above said optimized saponification conditions the average cholesterol content estimated using EDK method in butter, kalakand, khoa and paneer was 277.72, 76.34, 95.22 and 79.57 mg/100 g product, respectively. Developed EDK method can be used as a routine method to test the cholesterol content in indigenous dairy products to measure the batch to batch variation.

Aegle marmelos Leaf Extract is an Effective Herbal Remedy in Reducing Hyperglycemic Condition: A Pre-clinical Study

Diabetes is one of the alarming health problem in Pakistan. According to international diabetes federation (IDF), about 7 million Pakistanis are victims of diabetes accounting a total of 3% population. Most of Pakistanis still prefer to use herbal medicines for almost all of their health issues. Aegle marmelos (commonly known as the bael tree) is highly reputed ayurvedic medicinal tree as it has been used in traditional medicine for centuries. In this pre-clinical work, 32 mice were induced for diabetes via injecting alloxan intraperitoneally at a dose of 100 mg/kg body weight and mice having 104-170 mg/dl sugar level were considered diabetic. All mice were divided into two groups, each group with three sub-groups containing normal, control and diabetic mice. One group was given normal feed whereas other group was given a food containing Aegle marmelos leaf extract at 300 mg/kg body weight as an effective dose against alloxan induced hyperglycemia. The experiments lasted for 60 days and blood samples (05 ml to 1.0 ml) were collected from coccygeal vein of rats on 1st day, 30th day and 60th day of the experiment in heparinized tubes and blood glucose level was measured using spectrophotometer by enzymatic kit at a wavelength of 540nm. It was observed that the Aegle marmelos leaf extract is an effective herbal remedy in reducing and maintaining the glucose level in normal and hyperglycemic mice

Use of dried blood spot as an alternative method to estimate serum triglyceride in field studies

Introduction: Cardiovascular diseases are showing an increasing trend in both developing as well as developed countries. Raised triglycerides is considered as an important risk factor of cardio vascular diseases. Collection, storage and transportation of the blood samples from the field becomes a major limiting factor in areas lacking modern laboratory facilities. In such situations, dry blood or serum spots can become an alternative for the estimation of triglyceride. The objectives of our study was to evaluate the use of dry blood or dry serum spot to estimate triglyceride levels as an alternative method to estimate serum triglyceride and also to study the effect of storage time and storage temperature on the triglyceride content in the dry blood and serum spots. Methodology: Blood samples were collected from 100 study participants. Seven spots each of blood and serum from these samples were made on Whatman paper 3. After drying, one dried spot each from blood and serum was analysed on same day. Three of blood and three of serum dried spots were kept at room temperature and remaining at a temperature of 2-8° in separate re-sealable bags which were analysed on days 7, 15 and 30. Triglyceride was measured by Glycerol-3-phosphate oxidase-Peroxidase method. For estimating triglycerides in the eluate, 100 microlitre of the extract from the test-tube was added to 1 ml of the commercially available enzymatic reagent kit and incubated at 37°C for 15 min, and the absorbance was measured at 500 nm using a spectrophotometer. The data collected was analysed by Pearson’s correlation using SPSSv15. Results: Study showed a good correlation between the serum triglyceride values and the dry serum or blood spot triglyceride values. Dried serum spot triglyceride was found to be stable for about 15 days at room temperature and 7 days at 2-8°C with good correlation with serum value. The triglyceride concentration in the dry blood spots remained stable for 7 days at both room temperature and at 2-8°C. Conclusion: Dry blood/serum spots can be used as an alternative to serum for the estimation of triglyceride level which can be used to estimate various biochemical parameters at community level in remote areas which lack modern laboratory facilities. Key words: Serum triglycerides, Dry blood spot, Dry serum spot, cardiovascular diseases, surveillance

Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria

Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria

Relationship of dyslipidemia, insulin resistance, and prostate-specific antigen with prostate cancer

Background and Objective: The incidence of prostate cancer is increasing day by day worldwide. Prostate cancer in India is the 10 th most common malignancy affecting men although its incidence is rising in India. This study is designed to the effect of dyslipidemia, altered serum glucose, insulin resistance, and prostate-specific antigen (PSA) on the risk of prostate cancer. Materials and Methods: The study was conducted on a total of 150 patients, in which 75 patients were of prostate cancer considered as cases and 75 were healthy individuals as controls. About 8 ml of blood samples was drawn to determine fasting glucose, lipid profile, serum insulin, and serum PSA. Serum glucose, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and triglyceride (TG) were measured using enzymatic kits of auto analyzer. Very low-density lipoprotein-C (VLDL-C) and LDL-C were calculated by Friedwald's formula. Serum insulin and serum PSA were estimated by immunoenzymatic assay. Body mass index (BMI) was calculated as weight in kilograms divided by height in meters squared (kg/m 2 ). Insulin resistance was assessed by homeostasis model assessment insulin resistance index (HOMA-IR) and calculated as follows: fasting glucose (mg/dL) × fasting insulin (mU/mL)/405. Blood pressure was measured in the sitting position after a 10 min resting period. Observation and Results: Clinical variables such as age, BMI, blood pressure, lipid profile, serum glucose, serum insulin, HOMA-IR, and serum PSA in case and control groups were compared using the unpaired Student's t -test. We found that BMI and the level of serum glucose, serum insulin, HOMA-IR, and serum PSA were significantly increased in prostate cancer patients as compared to control. In prostate cancer patients, HDL-C significantly decreased ( P P Conclusion: This study has shown significant association of high BMI, dyslipidemia, insulin resistance, and PSA with prostate cancer.

Effect of Hormonal Contraceptives on the Total Antioxidants Status of Women from Isolo, Lagos State, Nigeria

The use of hormonal contraceptives has been implicated in the depletion of serum antioxidants and resultant promotion of oxidative stress which is associated with various disorders including cardiovascular disease and cancer. This study investigated serum total antioxidant status (TAS) in women taking hormonal contraceptives and compared their results with non-contraceptive users (age- and sex-matched). Sixty women aged 30 - 45 years, were enrolled for the study which included forty-five users of hormonal contraceptives from the Planned Parenthood Federation of Nigeria, Isolo, Lagos state and fifteen age-matched, apparently healthy, non-contraceptive users from the same geographical location were selected as controls. The in vitro determination of the serum total antioxidant status (TAS) was performed using Biorex enzymatic kit. Data obtained were statistically analyzed using student’s t-test and P < 0.05 was considered significant. The serum levels of TAS in the users of hormonal contraceptives were significantly lower than non-users (P < 0.05). The study concluded that women on hormonal contraceptives especially those taking either combined oral or combined injectable contraceptives were at risk of developing disorders associated with reduced levels of serum antioxidants.

Apolipoprotein B Is a Good Tool for Screening Dyslipidaemia in Apparently Healthy Population

Dyslipidaemia is the major risk factor for cardiovascular disease (CVD) which is the leading cause of death in the world. Even though several lipid parameters are used, currently apolipoprotein B (apoB) is considered as the best predictor of CVD. Thus this study was carried out to find out the association between conventional lipid parameters and apoB in apparently healthy subjects. A descriptive cross-sectional study was carried out in 170 apparently healthy volunteers who were not diagnosed with dyslipidaemia. After 12 hours overnight fast venous blood was obtained and Total cholesterol (TC), Triglyceride (TG), High density lipoprotein cholesterol (HDL-C) were measured by enzymatic kit method. Low density lipoprotein cholesterol (LDL-C) was calculated by Friedewald formula and apoB was analyzed by immune turbid metry using a Konelab® auto analyzer. Among the participants, majority (63.5%) were females. The mean value of apoB concentration of the population was 103 ± 42 mg/dL which was similar and not significantly different between the genders (Males, 102 ± 37 mg/dL and Females, 104 ± 45 mg/dL). All lipid parameters showed a positive correlation with apoB concentration whereas HDL-C had a negative correlation (r = -0.165). HDL-C significantly (p < 0.05) decreased with increase in apoB concentration while LDL-C, TC/HDL-C and non-HDL-C significantly (p < 0.05) increased with an increase in apoB concentration. Present study suggests that serum apoB has better correlations and associations with the parameters that are used in conventional lipid profile and with markers recommended for diagnosing dyslipidaemia. Hence apoB could be used as a single marker for screening dyslipidaemia in apparently healthy people.

PURIFIKASI PARSIAL DAN KARAKTERISASI ß-GALAKTOSIDASE Lactobacillus plantarum B123 INDIGENOS DAN HIDROLISIS LAKTOSA UNTUK PRODUKSI SUSU ULTRA HIGH TEMPERATURE RENDAH LAKTOSA

β-Galactosidase is enzyme which hidrolyze lactose to glucose and galactose. This enzyme is used in production low lactose milk for consumption human which have lactose intolerance. Partial purification of β-galactosidase is important to be conducted to increase β-galactosidase activity in order to its hydrolysis potency on UHT milk lactose increased.This research was aimed to production by partially purification and characterization indigenous β-galactosidase from Lactobacillus plantarum B123, and lactose hydrolysis for production low lactose UHT milk. Partially purification were precipitation following dialysis. Characterization included optimazion and stabilization of enzyme, while lactose hydrolisis for production low lactose UHT milk was detected by enzymatic GOD-POD kit. The results showed that production of β-galactosidase by using partial purification increased from 21.51 ± 0.23 U/mL (crude) to 106.34 ± 0.56 U/mL (dialysis). The optimum crude β-galactosidase activity was reached in precipitation by using 60 % ammonium sulphate. The purity of crude β-galactosidase increased 3.71 times after precipitation, and 14.28 times after dialysis. Characterization of β-galactosidase showed that optimum activities of crude and dialyzed β-galactosidase were at pH 6.5 and 50 oC, respectively. Stability of crude β-galactosidase incubated for 1 h were at pH: 5.0-8.5 and 25-50 °C. Specific activity of crude β-galactosidase was 15.05 U/mg protein, while that dialyzed β-galactosidase was 109.58 U/mg protein. Lactose hidrolysis to produce low lactose UHT milk showed that glucose concentration increased with the increase of hidrolysis time. Time needed to hidrolyze lactose 50 % with 4.8 U/mL β-galactosidase at 50°C was 6.08 h. In conclusion that indigenous β-galactosidase from Lactobacillus plantarum B123 purified partially can be used as lactose hidrolyzer in production of low lactose UHT milk.Key words : b-galactosidase, indigenous Lactobacillus plantarum B123, purification, lactose hidrolysis, UHT Milk