Glycosaminoglycan-induced proinflammatory cytokine levels as disease marker in mucopolysaccharidosis.

Recently, it has been shown disturbances in oxidant/antioxidant system and increases in some inflammatory markers in animal studies and in some Mucopolysaccharidoses (MPSs) patients. In this study, we aimed to determine the oxidative stress/antioxidant parameters and pro-inflammatory cytokine levels in the serum of MPS patients, in order to evaluate the possible role of inflammation in these patient groups regarding to accumulated metabolites. MPS I (n = 3), MPS II (n = 8), MPS III (n = 4), MPS IVA (n = 3), MPS VI (n = 3), and VII (n = 1) patients and 20 age-matched healthy subjects were included into the study. There was no statistically significant change in activities of SOD, Catalase, GSH-Px and lipid peroxidation levels in erythrocytes between the MPS patients and healthy controls. While IL-1alpha (p = 0.054), IL-6 (p = 0.008) levels, and chitotriosidase activity (p = 0.003) elevated in MPS3 patients, IL1α (p = 0.006), IL-1ß (p = 0.006), IL-6 (p = 0.006), IFNγ (p = 0.006), and NFκB (p = 0.006) levels increased in MPS-6 patients. Elevated levels of IL-6, IL1α and chitotriosidase activity demonstrated macrophage activation in MPSIII untreated with enzyme replacement. Our study showed for the first time that high levels of IL1α, IL-6, IL1ß and NFκB were present in MPSVI patients, demonstrating the induction of inflammation by dermatan sulphate. The low level of paraoxonase in MPSVI patients may be a good marker for cardiac involvement. Overall, this study provides important insights into the relationship between lysosomal storage of glycosaminoglycan and inflammation in MPS patients. It highlights possible pathways for the increased release of inflammatory molecules and suggests new targets for the development of treatments.

Development, optimization and validation of LC-MS/MS method for the determination of DBS GALT enzyme activity.

Galactosemia is a carbohydrate metabolism disorder often caused by galactose-1-phosphate uridyl transferase (GALT) deficiency. Detecting GALT deficiency involves measuring intra-erythrocyte enzyme activity. We aimed to create a robust liquid chromatography-mass spectrometry (LC-MS/MS) method to assess GALT activity in dried blood spot (DBS) samples. We validated this method and compared it to the fluorometric approach. We investigated the impact of K2EDTA and lithium heparin tubes on enzyme activity to identify the best sample collection tube. We also assessed the reaction-stopping method. The developed approach employed [13C6]-galactose-1-phosphate as a substrate and UDP-N-acetylglycosamine as an internal standard (IS). The mean ± SD value for GALT activity of DBS samples was determined as 6.37 ± 1.96 µmol/gHb/hour. The linear range was 0.4-50 µM (2.4-310% of normal) in the DBS method. The % coefficient of variation (%CV) values were less than 15 for intra-day and inter-day repeatability studies. Over 90% recovery was achieved in recovery studies, and no ion suppression from matrix was detected. DBS samples were quite stable for 31 days under different storage conditions. Enzyme activity results reported as <3.5 U/g Hb by fluorometric method, were quantitatively determined for even very low concentrations by LC-MS/MS method.

Design of a multiwalled carbon nanotube-Nafion-cysteamine modified tyrosinase biosensor and its adaptation of dopamine determination.

In this work, a multiwalled carbon nanotube (MWCNT)-Nafion-cysteamine (CA) modified tyrosinase biosensor brings a new and original perspective to biosensor technology intended for the development of dopamine determination. Dopamine measurements were done at 0.2V with the amperometric method by the developed biosensor system. In addition, in this study dopamine determination was carried out by using the differential pulse voltammetry method between potentials of 0.4 and -0.15 V. In the optimization studies of the biosensor, some parameters such as optimal pH, optimal temperature, optimal enzyme amount, and effect of MWCNT concentration were investigated. Afterward, in the characterization studies, some parameters such as linearity and reproducibility were determined. In the reproducibility experiment, an average value of 1.026 µM, a standard deviation of ±0.03975, and a coefficient of variation of 3.8% were determined for a 1-µM dopamine concentration (n=15). Determination of dopamine was carried out in drug samples by the developed biosensor.

Development of a New Amperometric Biosensor for Measurement of Plasma Galactose Levels.

Galactosemia is an inherited disease that occurs as a result of insufficient or no synthesis of some enzymes (GALT, GALK, and GALE) in galactose metabolism. Failure to make an early diagnosis, especially in newborns, can lead to severe clinical and even fatal consequences. The aim of this study is to develop a biosensor for measuring free galactose in plasma. The immobilization components of the developed free galactose biosensor are screen printed carbon electrode (SCPE), Prussian blue (PB), chitosan (CHIT), Nafion (NAF), gold nanoparticle (GNP), and galactose oxidase (GaOX). The CHIT/GaOX/NAF-GNP/GaOX/CHIT-GNP/SCPE-PB electrode showed a sensitive amperometric response to detect galactose. While the surface characterization of the biosensor was performed with cyclic voltammetry and scanning electron microscopy, the optimization and performance characterizations were made by applying an amperometry technique. The amperometric operating potential for the free galactose biosensor was determined as -0.05 V. The linear detection range for the free galactose biosensor is between 0.025 and 10 mM. This range includes galactose levels in plasma of both healthy and patients. The percent coefficient of variation values calculated for intraday and interday repeatability of the developed biosensor are below 10%. The practical use of the biosensor, for which optimization and characterization studies were carried out, was tested in 10 healthy 11 patients with galactosemia, and the results were compared with the colorimetric method. In conclusion, the unique analytical properties and effortless preparation of the new galactose biosensor developed in this study make them serious candidates for point-of-care diagnostic testing.

LC-MS/MS measurement of leukocyte cystine; effect of preanalytic factors.

Cystinosis is an autosomal recessive disorder characterized by the accumulation of cystine in lysosomes, causing irreversible damage to organs, especially the kidneys. Intracellular leukocyte cystine concentrations are used to diagnose cystinosis and to monitor cysteamine treatment. The aim of this study was to develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method without derivatization capable of measuring leukocyte intracellular cystine concentrations. During development, the effects of using three different protein precipitation agents were evaluated in terms of sensitivity and the matrix effect, with 12% trichloroacetic acid providing the highest sensitivity. The effects of different blood collection tubes were also assessed in terms of recovery, matrix effect, and protein content. Compared to other methods, our method was quicker (run time of 3 min), was linear over the range 0.078-100 µM, and had lower limits of detection (0.0192 µM) and quantification (0.0582 µM). The intra-day and inter-day reproducibility %CVs were ≤10%. and the method had excellent recovery rates (94%-106%). Other parameters including matrix selectivity, injection carryover, leukocyte lysate stability were also validated and met the acceptance criterias of European Medicines Agency (EMA) Guideline. The assay was successfully applied to quantify cystine leukocyte concentration in healthy and cystinosis patients.

The decision-making levels of urine tetrasaccharide for the diagnosis of Pompe disease in the Turkish population.

Background Recently, urinary excretion of the tetrasaccharide 6-α-D-glucopyranosyl-maltotriose (Glc4) has been proposed as a marker for the diagnosis and monitoring of Pompe disease (PD). We aimed to determine the reference intervals and reliable decision-making levels of urine tetrasaccharide concentrations for the diagnosis of infantile- and late-onset Pompe patients in the Turkish population. Methods In this study, nine patients with PD (five of them with late-onset PD [LOPD]) and 226 healthy individuals (aged 0-64 years) were included. Urine Glc4 concentrations were determined using the ultra-high-performance liquid chromatography (UHPLC) tandem mass spectrometry (MS/MS) method. Results Our data showed that the urine tetrasaccharide levels decreased with age in healthy individuals (p < 0.001, r = -0.256). It was higher especially during the first year of life compared to that in the elder subjects. The tetrasaccharide level of Pompe patients was higher compared to that of healthy controls of the same age: 99 ± 68 mmol/mol creatinine for infantile onset vs. 4.0 ± 3.0 mmol/mol creatinine for healthy controls of the same age group and 12.1 ± 17.4 mmol/mol creatinine for late onset vs. 1.7±1.2 mmol/mol creatinine for healthy controls of the same age group. Conclusions The results of this study showed that the reference intervals of tetrasaccharide in urine changed over time; therefore, it is critically important to define age-based decision levels for the diagnosis of LOPD.

Non-invasive cortisol detection in saliva by using molecularly cortisol imprinted fullerene-acrylamide modified screen printed electrodes.

In this study, we developed an impedimetric sensor system by using molecularly cortisol imprinted acrylamide polymers on fullerene modified carbon electrode to detect cortisol in real saliva samples. The polymer layer was formed on fullerene modified screen printed carbon electrodes with the ratio of 1:2 (cortisol:acrylamide) and ammonium persulfate (APS) was used as the initiator of polymerization. The sensor surface was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and Scanning Electron Microscopy (SEM). Performances of the electrode were determined as follows; calibration curve was calculated between 0.5 nM and 64 nM, R2 = 0.9939, LOD and LOQ were 0.14 nM and 0.44 nM, respectively. Real human saliva samples were obtained from 15 participants between 25 and 32 ages regardless of gender. The samples, which were collected at least 30 min after waking up and the new sensor method analysis were compared tandem mass spectroscopy coupled liquid chromatography system (LC-MS/MS). The sensor analysis showed a significant correlation with LC-MS/MS results (R2 = 0.9727).

Biotransformation of propolis phenols by L. plantarum as a strategy for reduction of allergens.

Propolis is a natural product produced by honeybees. It has antioxidant effects as well as antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic properties. Except these important healthy properties some cytotoxic effects causing allergies also have been reported. In this study have been evaluated changes of phenolic compounds including allergens molecules found in propolis. Before biotransformation, propolis samples were treated with different solvent (ethanol and polyethylene glycol) to facilitate solvation of solid samples. Biotransformation was done by three different strains of Lactobacillus plantarum (10, 8014, ATCC). Results demonstrated the importance of used solvent/treatment for extraction procedure and strains of L. plantrum. The lowest values of main allergens were determined as 321 ng/mL for BCAFE, 320 ng/mL for 1.1 DMAECAFE and 8.02 ng/mL for CAPE. The study is the first work deal with evaluation of bioconversion of propolis by different L. plantarum strains and their effects on phenolic profile.

MWCNT-cysteamine-Nafion modified gold electrode based on myoglobin for determination of hydrogen peroxide and nitrite.

In this work, a novel amperometric biosensor of hydrogen peroxide (H2O2) was developed based on the immobilization of myoglobin (Mb) on the surface of the multi-walled carbon nanotube (MWCNT) -Nafion-cysteamine (CA) modified gold electrode (Au) and its electrocatalytic activity was used for the determination of nitrite (NO2(-)). In the optimization studies, the best MWCNT and myoglobin amount were investigated. It was discovered at the experiments for the optimization of the working conditions that the buffer at this study as 50.0mM, pH7.0 phosphate buffer (PBS) and working temperature as 30°C for the H2O2 biosensor. It was determined at the characterization studies on the biosensor that linear results are obtained between the ranges of 0.1µM to 70.0µM for H2O2 concentration and 1-250µM for NO2(-). The reproducibility of the biosensor was determined both H2O2 and nitrite. From the experiments, average value, standard deviation (SD) and coefficients of variation (CV%) were calculated to be 10.02±0.43µM, and 4.29% for 10.0µM H2O2 (n=6) and 52.0±2.1µM, and 3.89% for 50.0µM nitrite (n=8), respectively. At the same time the sample was analyzed for NO2(-) in drinking and mineral waters.

A microbial biosensor based on Lactobacillus delbruecki sp. bacterial cells for simultaneous determination of lactic and pyruvic acid.

The aim of this study was mainly to develop a microbial biosensor for the simultaneous determination of lactic acid and pyruvic acid. In developing biosensor, lyophilised Lactobacillus delbruecki sp. bacterial cells were immobilised with polypyrrole on a platin electrode surface using electropolymerization method. Lactate concentration was determined based on the differences in amperometric responses at cathodic peak (+0.2V) of potassium ferricyanide, whereas pyruvate concentration was determined using the differences at anodic peak (+0.1V). The response of biosensor showed linearity between 0.1 and 1.0mM for both of two substrates. Optimisation studies were carried out for amount of microorganism, pyrrole concentration, pH and temperature. In the characterisation studies, substrate specificity, interference effect of some substances on the biosensor response, and storage stability were established.