Photometric Assay Research Articles

A coupled photometric assay for characterization of S-adenosyl-l-homocysteine hydrolases in the physiological hydrolytic direction

S-Adenosyl-l-homocysteine hydrolases (SAHases) are important metabolic enzymes and their dysregulation is associated with some severe diseases. In vivo they catalyze the hydrolysis of S-adenosyl-l-homocysteine (SAH), the by-product of methylation reactions in various organisms. SAH is a potent inhibitor of methyltransferases, thus its removal from the equilibrium is an important requirement for methylation reactions. SAH hydrolysis is also the first step in the cellular regeneration process of the methyl donor S-adenosyl-l-methionine (SAM). However, in vitro the equilibrium lies towards the synthetic direction. To enable characterization of SAHases in the physiologically relevant direction, we have developed a coupled photometric assay that shifts the equilibrium towards hydrolysis by removing the product adenosine, using a high affinity adenosine kinase (AK). This converts adenosine to AMP and thereby forms equimolar amounts of ADP, which is phosphorylated by a pyruvate kinase (PK), in turn releasing pyruvate. The readout of the assay is the consumption of NADH during the lactate dehydrogenase (LDH) catalyzed reduction of pyruvate to lactic acid. The applicability of the assay is showcased for the determination of the kinetic constants of an SAHase from Bradyrhizobium elkanii (KM,SAH 41±5μM, vmax,SAH 25±1μM/min with 0.13mg/mL enzyme). This assay is a valuable tool for in vitro characterization of SAHases with biotechnological potential, and for monitoring SAHase activity in diagnostics.

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis.We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An “IronChip” microarray analysis followed by a protein-protein interaction analysis was performed.FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis.The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported.

Photometric determination of free cholesterol via cholesterol oxidase and carbon nanotube supported Prussian blue as a peroxidase mimic

The authors describe a photometric method for the determination of free cholesterol based on the oxidation of cholesterol by the catalytic action of the enzyme cholesterol oxidase. The hydrogen peroxide formed is used to oxidize the chromogenic compound 3,3′,5,5′-tetramethylbenzidine (TMB) by exploiting the peroxidase-like activity of carbon nanotube-supported Prussian blue (PB). This enzyme mimic was synthesized from a mixture of ferric chloride and hexacyanoferrate(III) in the presence of intrinsically reducing multi-walled carbon nanotubes. The composite can trigger the formation of a blue-green dye from TMB in the presence of H2O2. The findings were exploited to design a photometric (652 nm) assay that has a linear response in the 4 to 100 μM cholesterol concentration range and a 3 μM detection limit. The practicability of the method was verified by the successful analysis of free cholesterol in human blood samples.

Torque Teno Virus (TTV) Among β-Thalassemia and Haemodialysis Patients in Mazandaran Province (North of Iran).

Torque Teno virus (TTV) is a transfusion transmitted virus that seems to be involved in several complications such as acute respiratory diseases, liver diseases, AIDS, cancer, and autoimmune reactions. In the present study the frequency of TTV was investigated among β- thalassemia (BT) and haemodialysis (HD) patients (high risk patients for TTV) in Mazandaran province, Iran. DNA was extracted from the serum of 82 BT and 100 HD patients, and nested PCR method was applied to detect TTV DNA. The aspartate transaminase(AST) and alanine transaminase (ALT) enzyme levels in BT patients were measured using photometric assay. The mean age of BT and HD patients as 23.4±5.4 and 48.8±8.2 years, respectively. 21% of HD and 26.8% of BT patients were infected with TTV, respectively. The frequency of TTV was not significantly different between two groups of patients and there was no significant correlation between sex and TTV infection. The mean AST and ALT levels in TTV positive BT patients was not significantly higher than TTV negative cases. The present study showed that TTV prevalence in BT patients with recurrent blood transfusion was not significantly higher than HD patients. The investigation of TTV prevalence in healthy individuals is recommended to identify if transfusion or dialysis is associated with higher TTV infection. Besides, although TTV infection did not change the AST and ALT enzymes in BT patients, the liver involvement may still exist in these patients.

Visual and colorimetric detection of ethylene glycol based on freeze-thawing induced aggregation of silver nanoparticles

The authors report on a simple colorimetric method for the determination of ethylene glycol (EG) in aqueous solution. It is based on the finding that EG prevents the aggregation of silver nanoparticles (AgNPs) on freezing-thawing. In the absence of EG, AgNPs aggregate after freeze-thawing because their surface were disrupted by ice crystals. This causes the solution to become colorless. However, in the presence of EG, ice crystals are not formed, and aggregation and decoloration do not occur. Under optimized conditions, the photometric assay, best performed at a wavelength of 395 nm, works in the 10 to 400 mM EG concentration range, and the detection limit is 3 mM (at a signal-to-noise ratio of 3). Concentrations down to 100 mM can be even detected visually.

A comparison of colorimetric assays detecting hydrogen peroxide in leaf extracts

Hydrogen peroxide (H2O2) is a central molecule in plant stress responses as a potential oxidizing agent or a signal molecule, depending on its localization and cellular concentrations. The work compares the versatility of three simple and rapid, potentially high through-put photometric assays to detect this reactive oxygen species in leaf extracts.

P2.01-088 Prenylation Inhibitors in Lung Adenocarcinoma: Comparison of Zoledronic Acid and a Novel Lipophilic Bisphosphonate

The prenylation inhibitor zoledronic acid is a standard-of-care therapeutic option in bone metastasis. Recent studies suggest that prenylation inhibition using novel lipophilic bisphosphonates might be active against various malignancies outside the bone metastatic setting. Since prenylation is an important posttranslational modification in RAS protein function we explored the sensitivity of a panel of lung adenocarcinoma cells representing various oncogenic driver mutations. 8 human lung adenocarcinoma cell lines were investigated in vitro to assess the short-term viability and long-term clonogenic potential following zoledronic acid and BPH-1222 treatments. The eight lung cancer cell lines represented wild type (HCC78, CALU3), EGFR- (H1650, H1975) KRAS- (A549, H358), BRAF- (CRL5885) and BRAF + NRAS double mutant (CRL5922) molecular subtypes. Effect on short and long term proliferation were measured with a SRB based photometric assay. Neither short-term nor long-term treatment showed significant differences between the proliferation inhibitory effect of the hydrophilic zoledronic acid and novel lipophilic bisphosphonate BPH-1222. Interestingly, we found that the two KRAS mutant lung adenocarcinoma cell lines were more sensitive in the long-term bisphosphonate treatment assays than non-KRAS mutant cell lines. BRAF or EGFR mutations did not show a differential response against these inhibitors. In vitro proliferation inhibitory efficacies of hydrophilic and lipophilic bisphosphonates were not different in lung adenocarcinoma cells. Nevertheless, due to the different bone accumulation properties of zoledronic acid and lipophilic bisphosphonates further in vivo preclinical studies are warranted to evaluate the inhibitory effect in a more physiological setting.

Photometric Assays for Chloroplast Movement Responses to Blue Light.

Assessment of chloroplast movements by measuring changes in leaf transmittance is discussed, with special reference to the conditions necessary for reliable estimation of blue light-activated chloroplast responses.

Enzyme activities in parotid saliva of patients with the restrictive type of anorexia nervosa

ObjectiveIn patients with anorexia nervosa (AN) specific signs may occur in the oral cavity, but there are conflicting reports about their significance, especially concerning changes in salivary composition.The aim of this clinical study was to evaluate the resting parotid flow rate (PFR) and the activity of the following enzymes in parotid saliva: amylase, aspartate amino transferase (AST), lysozyme, peroxidase, serine and acidic proteases in the acute phase of the restrictive type of AN and to compare the findings with those in healthy controls. DesignForty-one subjects participated (20 patients with AN, 21 matched healthy controls), parotid saliva was collected using a modified Lashley cap at rest. Enzyme activities were measured with fluorimetric and photometric assays. ResultsThe unstimulated PFR was significantly lower than in the controls, lysozyme and AST activity was significantly lower, and amylase showed a high inter-individual variability. A positive correlation for amylase and lysozyme and negative ones for lysozyme and BMI, lysozyme and IBW%, serine protease and salivary flow were observed. ConclusionsThe reduced PFR and enzyme activities levels suggest that AN does not only affect the quantity of the saliva but also its quality and, its biological functions. The results obtained should help to provide a better understanding of the effect of AN disease on the pathogenesis of at least some oral diseases. Further research is needed on any possible role of reduced lysozyme and transaminase activity in maintaining oral protection against external toxic agents and bacteria.

Hyperlipidemia-Mediated Increased Advanced Lipoxidation End Products Formation, an Important Factor Associated with Decreased Erythrocyte Glucose-6-Phosphate Dehydrogenase Activity in Mild Nonproliferative Diabetic Retinopathy.

The present study aimed to evaluate the role of hyperlipidemia in increased formation of advanced lipoxidation end products (ALEs) and to evaluate whether there is any relationship between ALEs generation and erythrocyte glucose-6-phosphate dehydrogenase (G6PD) activity in cases of mild nonproliferative diabetic retinopathy (MNPDR). In this study, we enrolled 100 patients with type 2 diabetes and MNPDR, 100 subjects with type 2 diabetes but without retinopathy (DNR) and 90 normal individuals without diabetes as healthy controls (HCs). Erythrocyte nicotinamide dinucleotide phosphate (NADPH), G6PD activity, serum total cholesterol, low- and high-density lipoprotein (LDL, HDL) and triglyceride levels were determined by photometric assay. Serum malondialdehyde (MDA) protein adduct and hexanoyl-lysine (HEL) were measured by an enzyme-linked immunosorbent assay (ELISA). A robust linear relationship was observed between MDA protein adduct and LDL or cholesterol or triglyceride levels, and HEL and LDL or cholesterol or triglyceride levels in subjects with MNPDR (p=0.0001). A significant inverse association was observed between erythrocyte G6PD activity and serum MDA protein adductor HEL levels in subjects with MNPDR (p=0.0001). Hyperlipidemia is an important factor that is associated with increased ALEs formation in persons with MNPDR. Increased ALEs generation was associated with decreased G6PD activity and low NADPH levels in cases of MNPDR, suggesting their detrimental role in the occurrence of early NPDR.

Eulophia macrobulbon – an orchid with significant anti-inflammatory and antioxidant effect and anticancerogenic potential exerted by its root extract

BackgroundThe Orchidaceae family is one of the largest families of flowering plants. Orchids are widely used for the traditional herbal medicine, acting as aphrodisiac, antisepic, antimicrobial, anti-cancer agent, etc. PurposeThis study was designed to elucidate the anti-inflammatory, antioxidant and cytotoxic potential of a 50% ethanolic extract of Eulophia macrobulbon roots (EME) in vitro, an orchid growing in Southern Asia. Furthermore, the main active compounds were isolated, and the bioactivity of the single constituents was determined. MethodsThe anti-inflammatory activity of EME and its compounds was evaluated by the secretion of pro- and anti-inflammatory cytokines and by the expression of inducible nitric oxide synthase (iNOS) in a lipopolysaccharide (LPS)-stimulated macrophage model, as determined by an enzyme linked immunosorbent assay (ELISA) and Western blot. Antioxidant activity was assessed using a DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) photometric assay. Cytotoxic effects were determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. ResultsEME and its compounds significantly reduced the production of the proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α), the expression of iNOS and subsequently increased the production of the anti-inflammatory cytokine interleukin 10 (IL-10) in LPS-stimulated macrophages. Additionally it could be demonstrated that EME is rich in radical scavengers. Furthermore, EME and its components showed notable cytotoxic effects on the human cervical adenocarcinoma cell line HeLa, the human colorectal adenocarcinoma cell line CaCo-2 and the human breast adenocarcinoma cell line MCF-7. The most active constituents were identified as 4-methoxy-9,10-dihydro-2,7-phenanthrenediol (8), 4-methoxy-2,7-phenanthrenediol (9), 1,5-dimethoxy-2,7-phenanthrenediol (10), 1,5,7-trimethoxy-2,6-phenanthrenediol (11), 1-(4-hydroxybenzyl)-4,8-dimethoxy-2,7-phenanthrenediol (15). ConclusionBased on this data, EME provides various beneficial anti-inflammatory, antioxidant and cytotoxic attributes and may be used as herbal remedy in the pharmaceutical or food industries.

β-galactosidase Production by Aspergillus niger ATCC 9142 Using Inexpensive Substrates in Solid-State Fermentation: Optimization by Orthogonal Arrays Design.

Background:Enzymatic hydrolysis of lactose is one of the most important biotechnological processes in the food industry, which is accomplished by enzyme β-galactosidase (β-gal, β-D-galactoside galactohydrolase, EC 3.2.1.23), trivial called lactase. Orthogonal arrays design is an appropriate option for the optimization of biotechnological processes for the production of microbial enzymes.Methods:Design of experimental (DOE) methodology using Taguchi orthogonal array (OA) was employed to screen the most significant levels of parameters, including the solid substrates (wheat straw, rice straw, and peanut pod), the carbon/nitrogen (C/N) ratios, the incubation time, and the inducer. The level of β-gal production was measured by a photometric enzyme activity assay using the artificial substrate ortho-Nitrophenyl-β-D-galactopyranoside.Results:The results showed that C/N ratio (0.2% [w/v], incubation time (144 hour), and solid substrate (wheat straw) were the best conditions determined by the design of experiments using the Taguchi approach.Conclusion:Our finding showed that the use of rice straw and peanut pod, as solid-state substrates, led to 2.041-folds increase in the production of the enzyme, as compared to rice straw. In addition, the presence of an inducer did not have any significant impact on the enzyme production levels.

In Vitro Assay for the Rap GTPase-Activating Protein Activity of the Purified Cytoplasmic Domain of Plexin.

Plexins are cell surface receptors that bind semaphorins and regulate essential processes such as axon guidance and angiogenesis. The cytoplasmic regions of plexins contain a functionally essential GTPase-activating protein (GAP) domain, which initiates downstream signaling by specifically inactivating the Rap GTPase. Here we describe the methods for expression and purification of the plexin cytoplasmic region in E. coli, and characterization of its GAP activity using a photometric assay. We also provide a protocol for measuring GAP activity of single-chain constructs with Rap covalently linked to the plexin cytoplasmic region.

Aptamer based photometric assay for the antibiotic sulfadimethoxine based on the inhibition and reactivation of the peroxidase-like activity of gold nanoparticles

It is known that gold nanoparticles (AuNPs) possess peroxidase-like activity. They can catalyze the oxidation of 3,3,5,5-tetramethylbenzidine by H2O2 which leads to a color change from red to blue. It is shown here that the peroxidase-like activity of AuNPs can be inhibited by passivating its surface passivation with a ssDNA aptamer against sulfadimethoxine. If, however, the target molecule (sulfadimethoxine) is present, the aptamer is desorbed from the AuNPs surface, and this results in the reactivation of the catalytic property of the AuNPs. The color change of the solution (from purple to blue) is related to the analyte concentration, and this can be judged visually or by UV-visible absorptiometry at 650 nm. The assay, under optimized conditions, has a detection limit of 10 ng·mL−1 of sulfadimethoxine, and the calibration plot is linear over a rather wide concentration range (0.01–1000 μg·mL−1). The assay can be performed within <15 min, is sensitive, and therefore is well suited for fast screening in food analysis. Conceivably, it can be extended to many other small analytes for which aptamers are available.

Constituents from oak bark (Quercus robur L.) inhibit degranulation and allergic mediator release from basophils and mast cells in vitro

Ethnopharmacological relevanceOak bark has been used since ancient times in Europaen ethnomedicine because of its adstringent, antimicrobial and hemostatic features, e.g. as a remedy for the treatment of wounds and skin diseases. PurposeOak bark tannins are considered as bioactive natural products, interacting with surface proteins of mucous membranes and might be beneficial for the treatment of allergic diseases. This study investigated the effect of an oak bark decoction (OBD) and isolated tannin fractions on the degranulation capacity and cytokine/chemokine release from rat basophilic cells and human mast cells in vitro, which are essential for the initiation of early- and late-phase allergic reactions. Methods and methodsBy chromatographic separation on Sephadex® LH-20 high- and low-molecular weight tannins were separated from OBD and the tannin composition analyzed by HPLC(DAD)-MSn. Then, the OBD and its fractions were tested in degranulation (β-hexosaminidase activity) of allergen-specific-activated basophilic cells in a photometric assay. ResultsThe OBD and the high-molecular tannin fraction showed a dose-dependent inhibition of cell degranulation. Furthermore, the OBD and particularly its high molecular weight tannin fraction exhibited an inhibitory activity on the IL-8-, IL-6- and TNF-α-secretion from stimulated human mast cells, detected and quantified by ELISA. ConclusionThe OBD and its high-molecular weight tannins revealed an impact on allergic mediator release of basophilic cells and human mast cells and thereby provide a rationale for the topical treatment with OBD preparations.

Colorimetric detection of H2O2 using flower-like Fe2(MoO4)3 microparticles as a peroxidase mimic

The authors describe the preparation of Fe2(MoO4)3 materials with micrometer dimensions, and show that they are highly efficient peroxidase mimics that can be used in a photometric assay for H2O2. Depending on the experimental conditions of synthesis, the Fe2(MoO4)3 micromaterials display different morphologies, surface properties, and peroxidase-like activities. Among them, Fe2(MoO4)3 with flower-like structure has the largest BET specific surface area and the most negative potential. It exhibits the best peroxidase mimetic activity in catalyzing the oxidation of 3,3′,5,5′-tetramethylbenzidine in the presence of H2O2 to generate a blue-green product. Based on these findings, method was worked out for H2O2 determination that allows both visual and photometric read-out. The assay has a detection limit as low as 0.7 μM, is selective and practical. Given its sensitivity, the method has a good potential with respect to determination of H2O2 in clinical samples but also in oxidase-type of reactions where H2O2 is being produced.

Preparation of novel ring-A fused azole derivatives of betulin and evaluation of their cytotoxicity

An efficient scheme to synthesize novel ring-A fused heterocyclic derivatives of betulin was developed. The starting reaction of this synthesis was one-pot selective bacterial oxidation of betulin to betulone used as the key compound to synthesize the substituted azoles such as C(2)–C(3)-fused 1,2,3-triazoles, oxazoles and 1,2,4-triazine, as well as C(1)–C(2)-fused isoxazoles. The semi-synthetic compounds were screened for their cytotoxic activity against human cancer cell lines A549, HCT 116, HEp-2, MS and RD TE32 with use of the photometric MTT assays. Among the tested compounds, N-acetyltriazole of betulin (10) displayed impressive cytotoxic activity with IC50 2.3–7.5 μM against HCT 116, HEp-2, MS and RD TE32 cell lines as well as 3-methyl-4-oxido-1,2,4-triazine-derivative of betulonic acid (12) that was active against HCT 116 and HEp-2 cell lines with IC50 1.4 and 1.5 μM, respectively. Comparative experiments showed triazole (10) to have a lower cytotoxicity to normal epithelial cells, in comparison with compound (12). In accord with the in vivo acute toxicity test, the LD50 of triazole (10) exceeded 600 mg/kg. The ability of the most potent active triazole (10) to trigger apoptotic cell death was explored in the Annexin V-FITC test and by analyzing of caspase activity and morphological alterations in mitochondria and nuclei of HCT 116 cells.

A plant oxysterol, 28-homobrassinolide binds HMGCoA reductase catalytic cleft: stereoselective avidity affects enzyme function.

Understanding the influence of ubiquitously present plant steroids on mammalian cell biology is currently of interest. Feedback inhibition of HMGCoA reductase (HMGCR) catalytic activity in the transformation of HMG-CoA to mevalonate is a significant regulatory step in sterol biosynthetic pathway. To assess the role of dietary steroids in this biochemical transformation, the phytosteroid isoform 28-homobrassinolide (28-HB), 90% pure, obtained from Godrej Agrovet (India) was used to determine its effect on mammalian HMG-CoA reductase. Photometric assay of pure human and select rat tissue HMGCR post 28-HB oral feed, PCR-HMGCR gene expression, and in silico docking of 28-HB and HMGCoA on HMGCR protein template were carried out. Using an oral feed regimen of pure 28-HB, we noted a decrease of 16% in liver, 17.1% in kidney and 9.3% in testicular HMGCR enzyme activity, 25% in HMGCR gene expression and 44% in the activity of pure human HMGCR due to this plant oxysterol. In silico docking studies yielded binding metrics for 28-HB-HMGCR lower than for HMGCoA-HMGCR, indicating stronger binding of HMGCR by this ligand. 28-HB exerts differential effects on rat tissue HMGCR, down regulates liver HMGCR gene expression and significantly inhibits HMGCR activity.

A product of immunoreactive trypsinogen and pancreatitis-associated protein as second-tier strategy in cystic fibrosis newborn screening

BackgroundIn cystic fibrosis newborn screening (CFNBS), immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) can be used as screening parameters. We evaluated the IRT×PAP product as second-tier parameter in CFNBS in newborns with elevated IRT. MethodsData on 410,111 screened newborns including 78 patients with classical cystic fibrosis (CF) from two European centers were retrospectively analyzed by discrimination analysis to identify a screening protocol with optimal cutoffs. We also studied differences in PAP measurement methods and the association of IRT and PAP with age. ResultsPAP values differed systematically between fluorometric and photometric assays. The IRT×PAP product showed better discrimination for classical CF than PAP only as second-tier screening parameter (p<0.001). In CF patients, IRT decreased while PAP values remained high over years. In newborns without CF, IRT decreased after birth over weeks while PAP increased within days. ConclusionsThe IRT×PAP product performs well as second-tier cutoff parameter for CFNBS. Screening quality parameters depend on the analytic method and on age at blood collection.

Antioxidant, anti-inflammatory and antinociceptive activities of methanolic extract of Justicia secunda Vahl leaf

Background of studyPlants used for traditional medicine contain a wide range of substances which can be used to treat various infectious diseases. AimThe study evaluated the in vitro antioxidant, antinociceptive, and anti-inflammatory activities of the methanolic extract of Justicia secunda Vahl leaf. MethodsThe acute toxicity was performed with up and down method and the highest dose used was 2g/kg. The anti-inflammatory activity was evaluated using the carrageenan and formalin-induced paw edema models, and antinociceptive activity was evaluated using acetic acid-induced writhing reflex and tail flick test models while the antioxidant activity was evaluated using 2,2-diphenyl-2-picryl hydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) photometric assay. ResultsThe extract was well tolerated as no signs of toxicity or death were noticed during the period of observation. The extract produced a concentration dependent increase in antioxidant activities in both DPPH and FRAP models. The extract produced its optimum activity at 400μg/ml in both DPPH (54.07%) assay and FRAP (1.58μM) assay. The extract produced significant (P<0.05) dose-dependent increase in both anti-inflammatory and antinociceptive activities. The antinociceptive and anti-inflammatory activities of the extract (0.4g/kg) were comparable with the reference drugs (aspirin and pentazocine) used in the study. ConclusionThis study suggests that J. secunda possesses anti-inflammatory, antinociceptive and antioxidant activities and also provide the pharmacological basis for its uses in traditional medicine for these purposes.