Homocysteine thiolactone and other sulfur-containing amino acid metabolites are associated with fibrin clot properties and the risk of ischemic stroke.

Homocysteine (Hcy) and Hcy-thiolactone (HTL) affect fibrin clot properties and are linked to cardiovascular disease. Factors that influence fibrin clot properties and stroke are not fully understood. To study sulfur-containing amino acid metabolites, fibrin clot lysis time (CLT) and maximum absorbance (Absmax) in relation to stroke, we analyzed plasma and urine from 191 stroke patients (45.0% women, age 68 ± 12 years) and 291 healthy individuals (59.7% women, age 50 ± 17 years). Plasma and urinary levels of sulfur-containing amino acid metabolites and fibrin clot properties were significantly different in stroke patients compared to healthy individuals. Fibrin CLT correlated with fibrin Absmax in healthy males (R2 = 0.439, P = 0.000), females (R2 = 0.245, P = 0.000), female stroke patients (R2 = 0.187, P = 0.000), but not in male stroke patients (R2 = 0.008, P = ns). Fibrin CLT correlated with age in healthy females but not males while fibrin Absmax correlated with age in both sexes; these correlations were absent in stroke patients. In multiple regression analysis in stroke patients, plasma (p)CysGly, pMet, and MTHFR A1298C polymorphism were associated with fibrin Absmax, while urinary (u)HTL, uCysGly, and pCysGly were significantly associated with fibrin CLT. In healthy individuals, uHTL and uGSH were significantly associated with fibrin Absmax, while pGSH, and CBS T833C 844ins68 polymorphism were associated with fibrin CLT. In logistic regression, uHTL, uHcy, pCysGly, pGSH, MTHFR C677T polymorphism, and Absmax were independently associated with stroke. Our findings suggest that HTL and other sulfur-containing amino acid metabolites influence fibrin clot properties and the risk of stroke.

Comprehensive studies on the development of HPLC-MS/MS and HPLC-FL based methods for routine determination of homocysteine thiolactone in human urine.

The report presents a new, robust, and reproducible liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) and HPLC-fluorescence (FL) based methods for the determination of urinary homocysteine thiolactone (HTL). In particular, a versatile sample preparation procedure was designed to purify urine samples, involving chloroform liquid-liquid extraction (LLE) of HTL and its re-extraction (re-LLE) with formic acid, prior to chromatographic analysis. In relation to HPLC-FL assay, the quantification of HTL additionally uses o-phthaldialdehyde (OPA) as the on-column derivatization agent, while HPLC-MS/MS assay employs homoserine lactone (HSL) as an internal standard (IS). The baseline separation of the analyte and IS (if applicable) is accomplished under hydrophilic interactions chromatography (HILIC) and reverse phase (RP)-HPLC conditions in the case of HPLC-MS/MS and HPLC-FL based method, respectively. The assays linearity was observed within 20-400 nmol/L for HTL in urine, covering the expected unknown analyte's concentration in study samples. The value of 20 nmol/L in urine was recognized as the limit of quantification (LOQ) for both methods. The assays were successfully applied to urine samples delivered by fifteen apparently healthy volunteers showing that they are suitable for screening of human urine.

Application of the HPLC-ELSD technique for the determination of major metabolites of ibuprofen and creatinine in human urine.

The report presents robust and high throughput methods, based on liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD), for the simultaneous determination of major metabolites of ibuprofen (IBU), namely 2-hydroxyibuprofen and carboxyibuprofen (method A) as well as creatinine (Crn) (method B) in human urine. The assays primarily involve straightforward sample purification. For both methods, the chromatographic separation of the analytes is achieved within 8 min at room temperature on Poroshell 120 SB-C18 (75 × 4.6 mm, 2.7 µm) column using gradient elution. The eluents consisted of 0.1% formic acid in water and acetonitrile (method A) or water and methanol (method B) delivered at a flow rate of 1 or 0.5 mL/min, respectively. In relation to metabolites of IBU, the assay linearity was observed within 0.06-0.5 g/L in urine, while the Crn assay linearity was demonstrated within 0.5-30 mmol/L in urine. The limit of quantification for IBU metabolites was determined to be 0.06 g/L, and 0.5 mmol/L for Crn. These methods were successfully applied to urine samples delivered by ten apparently healthy donors showing that the HPLC-ELSD assays are suitable for human urine screening.

One-pot sample preparation procedure for the determination of protein N-linked homocysteine by HPLC-FLD based method.

The report presents robust and high throughput method, based on liquid chromatography coupled with fluorescence detection (HPLC-FLD), for the determination of total protein N-linked homocysteine (Hcy) in human plasma. The assay involves simultaneous proteins precipitation with perchloric acid and removal of any other form of Hcy, except protein N-linked Hcy, via disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and plasma protein pellet wash with perchloric acid followed by liberation of N-linked Hcy from proteins by hydrochloric acid hydrolysis, drying under vacuum and residue reconstitution in diluted hydrochloric acid. The chromatographic separation of resulting in this way Hcy-thiolactone (HTL) is achieved within 3 min at room temperature on PolymerX RP-1 (150 × 4.6 mm, 5.0 µm) column using isocratic elution with eluent, consisted of o-phthaldialdehyde (OPA) in sodium hydroxide and acetonitrile (ACN), delivered at a flow rate 1 mL/min. The analyte is quantified by monitoring fluorescence at 480 nm using excitation at 370 nm, in a linear range from 0.25 to 10 µmol/L in plasma, while the limit of quantification (LOQ) equals 0.25 µmol/L. The method was successfully applied to plasma samples delivered by fifteen apparently healthy donors showing that the HPLC-FLD assay is suitable for screening of human plasma.

Simultaneous determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid and main plasma aminothiols by HPLC-UV based method.

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5'-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1-100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC-UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.

Plasma thiol levels and methylenetetrahydrofolate reductase gene c.665C > T and c.1286A > C variants affect fibrin clot properties in Polish venous thromboembolic patients.

BACKGROUND AND AIMS: Aminothiols, including cysteine (Cys) and glutathione (GSH) in relation to fibrin clot phenotype were not investigated in patients with venous thromboembolism (VTE) and 5,10-methylenetetrahydrofolate reductase (MTHFR) gene variants. We aimed to explore the associations between MTHFR variants and plasma oxidative stress indicators including aminothiols as well as fibrin clot properties with plasma oxidative status and fibrin clot properties in this group of patients. METHODS: In 387 VTE patients the MTHFR c.665C > T and c.1286A > C variants were genotyped, together with chromatographic separation of plasma thiols. We also determined nitrotyrosine levels and fibrin clot properties, including clot permeability (Ks), lysis time (CLT), and fibrin fibers thickness. RESULTS: There were 193 patients with MTHFR c.665C > T (49.9%) and 214 (55.3%) with c.1286A > C variants. Both allele carriers with total homocysteine (tHcy) levels >15 µM (n = 71, 18.3%), compared to patients with tHcy ≤15 µM had 11.5% and 12.5% higher Cys levels, 20.6% and 34.3% higher GSH levels as well as 28.1% and 57.4% increased nitrotyrosine levels, respectively (all P < 0.05). The MTHFR c.665C > T carriers with tHcy levels >15 µM compared to tHcy ≤15 µM had 39.4% reduced Ks and 9% reduced fibrin fibers thickness (both P < 0.05) with no differences in CLT. In the MTHFR c.1286A > C carriers with tHcy levels >15 µM, Ks was decreased by 44.5%, CLT prolonged by 46.1%, and fibrin fibers thickness was reduced by 14.5% compared to patients with tHcy ≤15 µM (all P < 0.05). Nitrotyrosine levels in MTHFR variants carriers correlated with Ks (r = -0.38, P < 0.05) and fibrin fibers diameter (r = -0.50, P < 0.05). CONCLUSIONS: Our study indicates that patients with MTHFR variants and tHcy >15 µM are characterized by elevated Cys and nitrotyrosine levels associated with prothrombotic fibrin clot properties.

Organic Acids Secreted by Lactobacillus spp. Isolated from Urine and Their Antimicrobial Activity against Uropathogenic Proteus mirabilis.

The natural microbiota of the urinary tract includes Lactobacillus spp., which secrete molecules with antimicrobial properties and have antagonistic activity against many pathogens. This paper focuses on the antibacterial effect of Lactobacillus strains isolated from urine against clinical strains of Proteus mirabilis isolated from kidney stones and from urine with coexisting urolithiasis. The study involved analyzing the main antimicrobial molecules secreted by Lactobacillus. In order to indicate which agent had the strongest antimicrobial effect, the supernatants were made alkaline and treated with catalase and high temperature. Both treated and untreated supernatants were analyzed for their activity. Exposing uropathogens to all untreated cell-free supernatants of Lactobacillus significantly reduced their growth, and it was established that these properties were related to organic acid secretion by these strains. Using LC-MS/MS and spectrophotometric techniques, lactic, citric, and succinic acids were determined qualitatively and quantitatively. The influence of these acids on the P. mirabilis growth and biofilm formation and their influence on membrane permeability were also investigated. The results indicate that organic acids secreted by Lactobacillus strains have a high antibacterial potential and could be used as novel agents in the treatment of urinary tract infections caused by P. mirabilis.

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine-Chromatographic Studies.

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography-mass spectrometry (GC-MS) based method was elaborated to identify and quantify TCA in human urine. The GC-MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1-50 µmol L-1 range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC-MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.

The Changes in Antioxidant Activity of Selected Flavonoids and Caffeine Depending on the Dosage and Form of Thiamine.

Phenolic compounds and thiamine may serve as therapies against oxidative stress-related neurodegenerative diseases. However, it is important to note that these components show high instability under changing conditions. The study's aim was to determine the impact of the thiamine concentration (hydrochloride-TH and pyrophosphate-TP; in the range 0.02 to 20 mg/100 g on the indices of the chelating properties and reducing power, and free radicals scavenging indices of EGCG, EGC, ECG and caffeine added from 0.04 to 6.0 mg/100 g. Our research confirmed that higher concentrations of TH and TP can exhibit significant activity against the test antioxidant indices of all components. When above 5.0 mg/100 g of thiamine was used, the radical scavenging abilities of the compound decreased in the following order: EGCG > ECG > EGC > caffeine. The highest correlation was found for the concentration of thiamine pyrophosphate to 20.0 mg/100 g and EGCG. Knowledge of the impact of factors associated with the concentration of both EGCG, EGC, ECG or caffeine and thiamine on their activity could carry weight in regulating the quality supplemented foods, especially of nutrition support for people of all ages were oral, enteral tube feeding and parenteral nutrition).

Microalgae-based removal of contaminants of emerging concern: Mechanisms in Chlorella vulgaris and mixed algal-bacterial cultures.

Incomplete removal of contaminants of emerging concern (CECs) has been reported for conventional wastewater treatment technologies. Microalgae-based treatment has recently gained interest thanks to simultaneous removal capacity of organic and inorganic compounds and potentially CECs. In this study, a lab-scale monoculture of Chlorella vulgaris and mixed microalgal-bacterial culture were compared in terms of removal of 28 CECs (bisphenols, 2018 EU Watch List substances, including neonicotinoids, pharmaceuticals, selected transformation products). The removal pathways in light and dark abiotic controls were also studied. Batch photobioreactors were run at hydraulic retention times of 11-12 days and CECs spiked at environmentally relevant concentrations (1-20 µg L-1). The mixed culture was better at removing bisphenols, compared to C. vulgaris. Bisphenols' log Kow was significant in removal pathways, where bisphenols with high log Kow were removed abiotically while bisphenols with low log Kow were mainly biodegraded. The removal degrees and the pathways of pharmaceuticals and EU Watch List substances were comparable between both cultures, showing no impact of log Kow for most compounds; however, the removal with C. vulgaris was faster for some. High log Kow was associated with high removal of estradiol in abiotic controls, showing the importance of adsorption onto biomass and suspended matter.

Pumpkin, Cauliflower and Broccoli as New Carriers of Thiamine Compounds for Food Fortification.

The aim of the study is to explore the possibility of vegetables being used as carriers of thiamine. The influence of carrier type (thiamine hydrochloride-TCh and thiamine pyrophosphate-TP) for the thiamine stability were investigated. Two varieties of pumpkin, Muscat and Hokkaido, as well as Cauliflower and Broccoli, were used as a matrix for the thiamine applied. The impregnated and freeze-dried vegetables were stored (230 days) with changing access to light (access to and restriction of light) and temperature (21 °C and 40 °C). The analyzed carriers were also used in the production of gnocchi dumplings. The content of thiamine was analyzed using the thiochromium method. In the study, consumer tests (n = 199) and sensory profiling were used to assess the impact of thiamine carriers on the sensory quality of gnocchi dumplings. It was found that the introduction of dried vegetables at the level of 30% allows for high sensory desirability of analyzed products, as well as suggesting the possibility of their frequent consumption. Such a product could potentially become an alternative to pork meat as a good source of thiamine. However, it should be noted that the thiamine losses may occur during the storage of dried vegetables and their culinary preparation.

Synergic Effect of Selected Ingredients and Calcium Chloride on the Technological, Molecular and Microbial Usefulness of Eggshells and Their Impact on Sensory Properties in a Food Model System.

Lower levels of calcium in adults increase the risk for osteoporosis, and in children, low calcium levels can impact their potential adult height. The study objective was to analyze the bioavailability and physicochemical properties of a calcium preparation based on chicken eggs. The base calcium preparation was enriched with one of a variety of biologically active substances, inter alia, vitamin D3, vitamin K, lysine, lactose, magnesium chloride and inulin. The newly developed calcium preparations were subjected to structural analysis using X-ray diffraction and scanning electron microscopy, and the hydrodynamic diameter for the molecules was determined using the dynamic light scattering method and their zeta potential. To determine the optimum storage conditions of calcium preparations, their hygroscopicity and bulk density were determined. The calcium preparations were also added to selected food products, such as apple juice with mango, fruit dessert (jelly) and beef meatballs. The enriched food products were subjected to sensory analysis. The study demonstrated the significant influence of additives to calcium preparation in terms of its hygroscopicity and morphology. It was found that all products with the addition of analyzed preparations were characterized by high sensory desirability. The results presented in the study comprise the basis for the development of new food products, enriched with calcium.

Gas Chromatography-Mass Spectrometry Based Approach for the Determination of Methionine-Related Sulfur-Containing Compounds in Human Saliva.

Gas chromatography-mass spectrometry technique (GC-MS) is mainly recognized as a tool of first choice when volatile compounds are determined. Here, we provide the credible evidence that its application in analysis can be extended to non-volatile sulfur-containing compounds, to which methionine (Met), homocysteine (Hcy), homocysteine thiolactone (HTL), and cysteine (Cys) belong. To prove this point, the first method, based on GC-MS, for the identification and quantification of Met-related compounds in human saliva, has been elaborated. The assay involves simultaneous disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP) and acetonitrile (MeCN) deproteinization, followed by preconcentration by drying under vacuum and treatment of the residue with a derivatizing mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA), and trimethylchlorosilane (TMCS). The validity of the method was demonstrated based upon US FDA recommendations. The assay linearity was observed over the range of 0.5-20 µmol L-1 for Met, Hcy, Cys, and 1-20 µmol L-1 for HTL in saliva. The limit of quantification (LOQ) equals 0.1 µmol L-1 for Met, Hcy, Cys, while its value for HTL was 0.05 µmol L-1. The method was successfully applied to saliva samples donated by apparently healthy volunteers (n = 10).

Quantification of homocysteine thiolactone in human saliva and urine by gas chromatography-mass spectrometry.

Homocysteine thiolactone (HTL) is a chemically reactive thioester that has been implicated in cardiovascular disease. So far, its presence has been documented in human and mouse plasma and urine. Here, using a new method, we show that HTL is present in human saliva. The assay involves chloroform-methanol extraction of HTL, lyophilization, and derivatization with N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method is based on a gas chromatography coupled with mass spectrometry (GC-MS) and quantifies HTL in a linear range from 0.05 to 1 µmol L-1 saliva and urine. The limit of quantification (LOQ) was 0.05 µmol L-1. With respect to saliva specimen, the accuracy was 98.7-112.6%, and 90.2-100.5%, while the precision was 7.1-13.5% and 12.5-15.0% for the intra- and inter-day variation, respectively. In relation to urine samples, the accuracy was 91.9-110.9% and 91.2-103.3%, while the precision varied from 2.2% to 14.5% and 7.4% to 14.3% for intra- and inter-day measurements, respectively. Using this method, we show that in apparently healthy individuals (n = 18), HTL levels in saliva are not positively correlated with urinary HTL levels. Undoubtedly, larger population should be investigated to get more meaningful results.

2-(3-Hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic Acid, Novel Metabolite of Pyridoxal 5'-Phosphate and Cysteine Is Present in Human Plasma-Chromatographic Investigations.

It is well-established that aminothiols, to which cysteine (Cys) belongs, are highly reactive towards aldehydes in an aqueous environment, forming substituted thiazolidine carboxylic acids. This report provides evidence that formation of the product containing a thiazolidine ring through non-enzymatic condensation of Cys and an active form of vitamin B6 pyridoxal 5'-phosphate (PLP) occurs in vivo in humans. To prove this point, a new method, based on a gas chromatography coupled with mass spectrometry (GC-MS), has been designed to identify and quantify Cys and PLP adduct, 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA) in human plasma. The GC-MS assay relies on sample deproteinization by ultrafiltration over cut-off membranes and preconcentration by drying under vacuum, followed by treatment of the residue with derivatization mixture containing anhydrous pyridine, N-trimethylsilyl-N-methyl trifluoroacetamide (MSTFA) and trimethylchlorosilane (TMCS). The method quantifies HPPTCA in a linear range from 1 to 20 µmol L-1, where the lowest standard on the calibration curve refers to the limit of quantification (LOQ). The validity of the method was demonstrated. Furthermore, the method was successfully applied to plasma samples donated by apparently healthy volunteers and breast cancer patients. The GC-MS assay provides a new tool that will hopefully facilitate studies on the role of HPPTCA in living systems.

Antioxidant potential of various solvent extract from Morus alba fruits and its major polyphenols composition / Potencial antioxidante de vários extratos de frutos de Morus alba e de sua composição majoritária de polifenóis

ABSTRACT: Extraction conditions are an important factor in the process of obtaining bioactive compounds from plant matrix. These compounds differ structurally. Structures of phyto-compounds and their interactions with other food ingredient are not fully known, while these two aspects should play a significant role in extrahents choice and determination of extraction process conditions. Mulberry (Morus alba) is a plant growing in Asia, which fruits are rich in bioactive ingredients and high anti-oxidative potential. In our study we analyzed mulberry fruits extracts differing in the extra hent applied: acetone, methanol, ethanol and water. All tested extracts possessed rich polyphenolic composition and radical scavenging ability. The significant differences among the extracts in phenolic acids and flavonoids compositions were noticed, where the highest values were observed for acetone extract. The extrahent applied affects the antioxidative profile of tested samples, as well. The highest scavenging activity against ABTS was observed for acetone and ethanol extracts, while the poorest activity had water extract. Similar results were provided for ferrous ion reducing test and Fe chlating activity (acetone>ethanol>methanol>water). These results are helpful when selecting solvents with appropriate bioactive compounds compositions and high phytochemical profiles to be used as ingredients in supplements, as well as in functional foods.

RESUMO: As condições de extração são um fator importante no processo de obtenção de compostos bioativos da matriz vegetal. Estes compostos diferem-se estruturalmente. As estruturas de fito conjugações e suas interações com outros ingredientes alimentares não são totalmente conhecidas, enquanto esses dois aspectos devem desempenhar um papel significativo na escolha de extrações e na determinação das condições do processo de extração. A amoreira (Morus alba) é uma planta que cresce na Ásia, cujos frutos são ricos em ingredientes bioativos e com alto potencial antioxidante. Em nosso estudo, analisamos extratos de frutos de amoreira diferindo no extraente aplicado: acetona, metanol, etanol e água. Todos os extratos testa dos possuíam composição polifenólica rica e capacidade de eliminação de radicais livres. As diferenças significativas entre os extratos em composições de ácidos fenólicos e flavonóides foram observadas, em que os maiores valores foram observados para o extrato de acetona. O extraente aplicado afeta também o perfil antioxidante das amostras testadas. A maior atividade de eliminação contra o ABTS foi observada para a acetone e etanol extração, enquanto a atividade mais pobre tinha água. Resultados semelhantes foram fornecidos para teste de redução de íons ferrosos e atividade de aglutinação de Fe (acetona>etanol>metanol>água). Estes resultados são úteis na seleção de solventes com composições apropriadas de compostos bioativos e altos perfis fitoquímicos para serem usados como ingredientes em suplementos, bem como em alimentos funcionais.

The Effect of Thiamine Concentration on the Antioxidative Activity Indices in Tea Extracts.

The aim of the study was to determine correlations between the concentration of thiamine in systems and indicators of the antioxidative activity of ethanol tea extracts in the presence of soybean oil. Variability of the thiamine form was assumed by comparison of the influence of thiamine hydrochloride or thiamine pyrophosphate and fermentation of ethanol tea extracts. The study provides practical knowledge about the antioxidative activity of ethanol tea extracts in products containing fat and thiamine. The study showed that all tea extracts exhibited higher antioxidative activity in the presence of thiamine amounts of 0.1 and 0.8 mg/100 g. The antioxidative activity of ethanol tea extracts was significantly reduced when the concentrations were higher than the natural level for foods (over 1.0 mg/100 g). The systems containing white tea extract were the most vulnerable, whereas those with black tea were the least vulnerable. The presence of thiamine pyrophosphate in the system was more strongly correlated with reduced activity of the extracts than the presence of thiamine hydrochloride.

Air-drying temperature changes the content of the phenolic acids and flavonols in white mulberry (Morus alba L.) leaves / Temperatura de secagem ao ar altera o teor de ácidos fenólicos e flavonóis em folhas de amoreira branca (Morus alba L.)

ABSTRACT: The white mulberry leaves are typically available on the market in dried or encapsulated form. It was assumed in the study that appropriate drying of leaves of the white mulberry is significant for obtaining intermediate products with high content of compounds having anti-oxidative activity. The purpose of the study was to determine the influence of the temperature of mulberry leaves air drying on the content of phenolic acids and flavonols. It has been determined that the content of these compounds in the leaves depended on the drying temperature. Drying at 60 °C favored release of phenolic acids and flavonols from complexes and/or formation of new compounds. Their total content was 22% higher than in leaves dried at 30 °C. Drying at 90 °C reduced the phenolic acid and flavonol content by 24%. The most favorable drying temperature was 60 °C.

RESUMO: As folhas da amoreira branca estão normalmente disponíveis no mercado em forma seca ou encapsulada. Assumiu-se no estudo que a secagem adequada das folhas da amora branca é importante para a obtenção de produtos intermediários com alto teor de compostos com atividade antioxidante. O objetivo do estudo foi determinar a influência da temperatura de secagem de ar de folhas de amoreira sobre o teor de ácidos fenólicos e flavonóis. Foi determinado que o conteúdo destes compostos nas folhas dependia da temperatura de secagem. Secagem a 60 °C favoreceu a liberação de ácidos fenólicos e flavonóis a partir de complexos e / ou formação de novos compostos. Seu teor total foi 22% superior ao das folhas secas a 30 °C. A secagem a 90 °C reduziu o teor de ácido fenólico e flavonol em 24%. A temperatura de secagem mais favorável foi de 60 °C.

Application of GC-MS technique for the determination of homocysteine thiolactone in human urine.

It is well established that homocysteine thiolactone (HTL) is associated with some health disorders, including cardiovascular diseases. HTL is a by-product of sulfur metabolic cycle. So far, its presence has been confirmed in human plasma and urine. It has been also shown that a vast majority of HTL is removed from human body through kidney. Thus, the aim of the current investigations has been the identification, separation and quantification of HTL in urine samples. For the first time a cheap, reliable and robust GC-MS method was developed for the determination of HTL in human urine in the form of its volatile isobutyl chloroformate derivative. Separation of the analyte and internal standard (homoserine lactone (HSL)) was achieved in 15 min followed by mass spectrometry detection (MS). Isocratic elution was accomplished with helium at a flow rate of 1 mL min-1 and a gradient of the column temperature was concomitant with the analysis. The mass spectrometer was set to the electron impact mode at 70 eV. The ion source, quadrupole and MS interface temperatures were set to 230 °C, 150 °C and 250 °C, respectively. Elaborated analytical procedure allows quantification of analyte in a linear range of 0.01-0.20 nmol mL-1 urine. The LOQ and LOD values were 0.01 and 0.005 nmol mL-1, respectively. The method accuracy ranged from 98.0% to 103.2%, while precision varied from 6.4% to 9.5% and from 10.7% to 16.9% for intra- and inter-day measurements, respectively. Finally, the method has been successfully implemented in the analysis of 12 urine samples donated by apparently healthy volunteers. Concentration of HTL ranged from

Antioxidant efficacy of Kalanchoe daigremontiana bufadienolide-rich fraction in blood plasma in vitro.

CONTEXT: The main source of bufadienolides is toad venom; however, plants such as members of Kalanchoe Adans. (Crassulaceae) genus may also synthesize these bioactive substances. OBJECTIVE: This is the first study on antioxidant effects and cytotoxicity of bufadienolide-rich fraction isolated from Kalanchoe daigremontiana Raym.-Hamet & H. Perrier. MATERIALS AND METHODS: The methanolic fraction was extracted from the plant roots and contained 0.48 mg bufadienolides/mg of dry mass (11α,19-dihydroksytelocinobufagin, bersaldegenin-1-acetate, bersaldegenin-1,3,5-orthoacetate, 19-(acetyloxy)-3ß,5ß,11α,14-tetrahydroxyl-12-oxo-bufa-20,22-dienolide and 19-(acetyloxy)-1ß,3ß,5ß,14-tetrahydroxyl-bufa-20,22-dienolide, mainly). The cytotoxicity of K. daigremontiana fraction was evaluated in an in vitro experimental model of blood platelets. The viability of blood platelets was determined on the basis of a release of lactate dehydrogenase. RESULTS: The fraction scavenged DPPH• radicals, with EC50 of 21.80 µg/mL. Studies on an experimental model of blood plasma under peroxynitrite-induced oxidative stress revealed that the plant preparation had moderate antioxidant properties. Levels of 3-nitrotyrosine and thiol groups indicated that the protective effect of K. daigremontiana was significant mainly for its concentration of 50 µg/mL. No effect was found in prevention of oxidation of low-molecular plasma thiols (glutathione, cysteine and cysteinylglycine). Simultaneously, measurements of lipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS) indicated that the examined fraction might be effective antioxidant at broader concentration range, that is 1-5 and 25-50 µg/mL for hydroperoxides and TBARS generation, respectively. No cytotoxicity was observed at the concentration range of 1-50 µg/mL. CONCLUSIONS: Based on the obtained results, we suggest that antioxidant activity may additionally contribute to beneficial properties of K. daigremontiana-derived extracts.