Isolation of Cathepsin Band a-N-Benzoylarginine-B-naphthylamide Hydrolase by Covalent Chromatography on Activated Thiol Sepharose

Cathepsin Band a-N-benzoylarginine-P,-naphthylamide (BANA) hydrolase have been isolated from bovine lymph nodes using a novel procedure that includes besides gel filtration and ion exchange chromatography also covalent chromatography as the essential step. Both enzymes were selectively bound to the activated thiol-Sepharose and afterwards eluted with cysteine. The homogeneity of enzymes was proved by polyacrylamide gel electrophoresis

Some Characteristics of Cathepsin Band a-N-Benzoylarginine- B-Naphthylamide Hydrolase From Bovine Lymph Nodes

Some properties of cathepsin B and a-N-benzoylarginine-~- naphthylamide (BANA) hydrolase from bovine lymph nodes have been studies. a-N-benzoylarginine-~-naphthylamide was a sensitive substrate for both enzymes. Leucine-2-naphthylamide was cleaved only by BANA hydrolase. Degradation of low molecular weight substrates was optimal at pH = 6.0. At this pH value, the enzymes were most stable. Cathepsin B inactivated aldolase, was inhibited by 1 μM leupeptin and by thiol blocking compounds. BANA hydrolase was not inhibited by 1 μM leupeptin but showed that it required thiol compounds and EDTA for full activation. It was concluded that BANA hydrolase is very similar or identical to cathepsin H from rat liver lysosomes

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014-2021)

As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6-12 years, (ii) 3,117 teenagers aged 12-18 years and (iii) 4,102 young adults aged 20-39 years. The participants were recruited between 2014 and 2021 in 11-12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Sigma (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures

Harmonized human biomonitoring in European children, teenagers and adults: EU-wide exposure data of 11 chemical substance groups from the HBM4EU Aligned Studies (2014–2021)

As one of the core elements of the European Human Biomonitoring Initiative (HBM4EU) a human biomonitoring (HBM) survey was conducted in 23 countries to generate EU-wide comparable HBM data. This survey has built on existing HBM capacity in Europe by aligning national or regional HBM studies, referred to as the HBM4EU Aligned Studies. The HBM4EU Aligned Studies included a total of 10,795 participants of three age groups: (i) 3,576 children aged 6–12 years, (ii) 3,117 teenagers aged 12–18 years and (iii) 4,102 young adults aged 20–39 years. The participants were recruited between 2014 and 2021 in 11–12 countries per age group, geographically distributed across Europe. Depending on the age group, internal exposure to phthalates and the substitute DINCH, halogenated and organophosphorus flame retardants, per- and polyfluoroalkyl substances (PFASs), cadmium, bisphenols, polycyclic aromatic hydrocarbons (PAHs), arsenic species, acrylamide, mycotoxins (deoxynivalenol (total DON)), benzophenones and selected pesticides was assessed by measuring substance specific biomarkers subjected to stringent quality control programs for chemical analysis. For substance groups analyzed in different age groups higher average exposure levels were observed in the youngest age group, i.e., phthalates/DINCH in children versus teenagers, acrylamide and pesticides in children versus adults, benzophenones in teenagers versus adults. Many biomarkers in teenagers and adults varied significantly according to educational attainment, with higher exposure levels of bisphenols, phthalates, benzophenones, PAHs and acrylamide in participants (from households) with lower educational attainment, while teenagers from households with higher educational attainment have higher exposure levels for PFASs and arsenic. In children, a social gradient was only observed for the non-specific pyrethroid metabolite 3-PBA and di-isodecyl phthalate (DiDP), with higher levels in children from households with higher educational attainment. Geographical variations were seen for all exposure biomarkers. For 15 biomarkers, the available health-based HBM guidance values were exceeded with highest exceedance rates for toxicologically relevant arsenic in teenagers (40%), 3-PBA in children (36%), and between 11 and 14% for total DON, Σ (PFOA + PFNA + PFHxS + PFOS), bisphenol S and cadmium. The infrastructure and harmonized approach succeeded in obtaining comparable European wide internal exposure data for a prioritized set of 11 chemical groups. These data serve as a reference for comparison at the global level, provide a baseline to compare the efficacy of the European Commission's chemical strategy for sustainability and will give leverage to national policy makers for the implementation of targeted measures

Encapsulation of natural antioxidant resveratrol in liposomes

Liposomes have been shown to be suitable systems for encapsulation and preserving the health-beneficial properties of a wide range of biological active ingredients such as resveratrol (RSV). The aim of this study was to encapsulate RSV in liposomes, with a goal to achieve the extended release and improved stability of RSV. Multilameral liposomes were prepared by means of two different methods: thin film method (TF) and proliposome method (PRO). In both methods, the ratio between added RSV and phospolipon 90G (P90G) was 1:20 w/w. Extrusion and sonication were applied in order to obtain unilameral liposomes. Both methods were efficient in capturing RSV within the microparticles, thus encapsulation efficiency had high values (92,9% in case of TF and 97,4% in case of PRO). The size reduction of liposomes resulted with particles of the average diameter ranged between 120 and 270 nm. Antioxidative activity was retained at a high level (approximately 95%). Franz diffusion cell was used for release studies and diffusion of RSV was monitored for 6h. According to the results, liposomes appeared to be suitable vehicles for encapsulation of resveratrol where PRO is particularly useful for encapsulation of antioxidants

Resveratrol loaded liposomes produced by different techniques

Several different methods for production of liposomes incorporating resveratrol were investigated and compared from the aspect of size distribution, surface charge, entrapment efficiency, phase behavior and stability. Thin film method and proliposome method provided high entrapment efficiency (92.9% and 97.4%, respectively). Extrusion and sonication techniques were applied to obtain particles of the average diameter between 120 and 270 nm. The sonicated liposomes incorporated resveratrol (44-56%) fewer than extruded vesicles (92-96%). Antioxidative activity of resveratrol was retained upon encapsulation. Differential scanning calorimetry was performed in order to study the interaction of liposomal membranes with resveratrol, and their physical state. The release studies performed in Franz diffusion cell showed that liposomes impart slow diffusion of resveratrol, where diffusion resistance derived from liposomal membrane ranged from 5.90 . 10(5) to 9.55 . 10(5) s/m depending on the size of particles. Cytotoxicity of the formulations was evaluated via morphological changes of keratinocytes treated by liposomes. Industrial Relevance: Resveratrol displays many health-beneficial properties and possesses a remarkably strong antioxidant activity. Although often consumed in food, the positive effects of resveratrol are restricted because it is prone to oxidation, poorly absorbed when orally administrated, and cytotoxic in higher total dosages (though relatively high local concentrations are required for an effect). Encapsulation is one way to improve bioavailability and stability of resveratrol; herein the main challenge is to find a suitable solution, as resveratrol is weakly water soluble. This has motivated us to design new formulations based on liposomes for delivering of resveratrol. In the food sector, liposomes have been investigated for delivering proteins, enzymes, antioxidants, flavors and vitamins. The mean advantage of liposomes over other encapsulation technologies (spray-drying, extrusion, and fluidized beds) is the stability that liposomes impart to water-soluble compounds in aqueous surroundings. Liposomes are able to stabilize the encapsulated materials against a range of environmental and chemical changes. Another important characteristic of liposomes is that, unlike many other existing encapsulants, they can be utilized in the entrapment, delivery, and release of poorly water soluble compounds, such as resveratrol, and they are also convenient for water-soluble, lipid-soluble, and amphiphilic compounds. As liposomes could be produced from naturally occurring components, regulatory issues that may prevent the application in food systems are potentially diminished, and new formulations could be quickly implemented. Despite benefits described here, up to date little use of liposomes in food systems has been made, as current manufacturing processes are mainly time consuming, often consisting of several steps with high costs of raw materials. Another problem is that devices available commercially which are utilized for production of liposomes are able to process only small quantities. Therefore, our research is devoted to the development of the process for liposome production which is easy to scale up, and at the same time, is effective as the common way based on thin film hydration process. The process elaborated in our study utilizes a commercial lipid mixture. The method used called proliposome method is based on replacement of ethanol solvent by aqueous media. For liposome downsizing, sonication (which can be easily modified to increase sample volume capability) is tested versus membrane extrusion (equipment for small-large batches is readily available). The goal of this article is to provide evidence for food manufacturers and food scientists to make broader use of resveratrol-loaded liposomes that can add value and improve the quality of existing food products