Comparative Analysis of Collagen-Containing Waste Biodegradation, Amino Acid, Peptide and Carbohydrate Composition of Hydrolysis Products

This paper aimed to study the biodegradation of collagen-containing waste (pork skin) induced by collagenase and Neutrase 1.5 MG enzymes and compare the amino acid, peptide, and carbohydrate composition of hydrolysis products. It was found that the degree of biodegradation of collagen-containing raw materials (pork skin) reached 78% when using an enzyme preparation (collagenase with a concentration of 250 U/g of the substrate) at pH 7.0, 40 °C, and a 360 min process duration. It was shown that the content of peptides with a molecular weight of 6.5–14.0 kDa in the hydrolysis products (collagenase) of collagen-containing wastes was 13.4 ± 0.40%, while in the products of hydrolysis (Neutrase 1.5 MG) it was 12.8 ± 0.38%. The study found that the hydrolysis products (Neutrase 1.5 MG) of collagen-containing raw materials contain fewer hexoses, free hexosamines, and hyaluronic acid than the hydrolysis products (collagenase) of collagen-containing raw materials. The content of chondroitin sulfates is practically the same in all samples of hydrolysis products. Proteases with collagenolytic activity are widely used in industry. Recently, they have increasingly been used in pharmaceutical, food, and other industries. Collagenases are promising enzymes for the production of chondroprotectors used for the treatment of osteoarthritis.

Increase in Chondroitin Sulfate and Decline in Arylsulfatase B May Contribute to Pathophysiology of COVID-19 Respiratory Failure

<b><i>Introduction:</i></b> The potential role of accumulation of chondroitin sulfates (CSs) in the pathobiology of COVID-19 has not been examined. Accumulation may occur by increased synthesis or by decline in activity of the enzyme arylsulfatase B (ARSB; N-acetylgalactosamine-4-sulfatase) which requires oxygen for activity. <b><i>Methods:</i></b> Immunostaining of lung tissue from 28 patients who died due to COVID-19 infection was performed for CS, ARSB, and carbohydrate sulfotransferase (CHST)15. Measurements of mRNA expression of CHST15 and CHST11, sulfotransferase activity, and total sulfated glycosaminoglycans (GAGs) were determined in human vascular smooth muscle cells following angiotensin (Ang) II treatment. <b><i>Results:</i></b> CS immunostaining showed increase in intensity and distribution, and immunostaining of ARSB was diminished in COVID-19 compared to normal lung tissue. CHST15 immunostaining was prominent in vascular smooth muscle cells associated with diffuse alveolar damage due to COVID-19 or other causes. Expression of CHST15 and CHST11 which are required for synthesis of CSE and chondroitin 4-sulfate, total sulfated GAGs, and sulfotransferase activity was significantly increased following AngII exposure in vascular smooth muscle cells. Expression of Interleukin-6 (IL-6), a mediator of cytokine storm in COVID-19, was inversely associated with ARSB expression. <b><i>Discussion/Conclusion:</i></b> Decline in ARSB and resulting increases in CS may contribute to the pathobiology of COVID-19, as IL-6 does. Increased expression of CHSTs following activation of Ang-converting enzyme 2 may lead to buildup of CSs.

Microbiological-Chemical Sourced Chondroitin Sulfates Protect Neuroblastoma SH-SY5Y Cells against Oxidative Stress and Are Suitable for Hydrogel-Based Controlled Release

Chondroitin sulfates (CS) are a class of sulfated glycosaminoglycans involved in many biological processes. Several studies reported their protective effect against neurodegenerative conditions like Alzheimer’s disease. CS are commonly derived from animal sources, but ethical concerns, the risk of contamination with animal proteins, and the difficulty in controlling the sulfation pattern have prompted research towards non-animal sources. Here we exploited two microbiological-chemical sourced CS (i.e., CS-A,C and CS-A,C,K,L) and Carbopol 974P NF/agarose semi-interpenetrating polymer networks (i.e., P.NaOH.0 and P.Ethanol.0) to set up a release system, and tested the neuroprotective role of released CS against H2O2-induced oxidative stress. After assessing that our CS (1–100 µM) require a 3 h pre-treatment for neuroprotection with SH-SY5Y cells, we evaluated whether the autoclave type (i.e., N- or B-type) affects hydrogel viscoelastic properties. We selected B-type autoclaves and repeated the study after loading CS (1 or 0.1 mg CS/0.5 mL gel). After loading 1 mg CS/0.5 mL gel, we evaluated CS release up to 7 days by 1,9-dimethylmethylene blue (DMMB) assay and verified the neuroprotective role of CS-A,C (1 µM) in the supernatants. We observed that CS-A,C exhibits a broader neuroprotective effect than CS-A,C,K,L. Moreover, sulfation pattern affects not only neuroprotection, but also drug release.

Dynamics of Change of Biochemical Indicators in Rats under the Influence of Sodium Glutamate

The purpose of the study was to determine metabolic parameters (alpha-amylase, alanine aminotransferase and aspartate aminotransferase, gamaglutamyl transpeptidase, glycoproteins and chondroitin sulfates) at the stages of sodium glutamate intake and after withdrawal. Materials and methods. Studies were conducted on 65 white male rats of reproductive age (2.5-3 months); the experimental group received 70 mg/kg of sodium glutamate per live weight for 8 weeks. The control group of animals received a standard diet. Before drug withdrawal, rats were studied every week, after drug withdrawal the studies were performed every two weeks (10, 12, 14, 16 weeks). Results and discussion. The study of the enzyme activity of alpha-amylase showed that this index increased on the average 1.3-fold compared with the control values, and at the end of the period when the rats were fed with sodium glutamate (8 weeks), the index increased by 3.85 times, alanine aminotransferase – by 12.3 times, aspartate aminotransferase – by 1.4 times, gamaglutamyl transpeptidase – by 2.7 times, glycoproteins – by 1.4 times, chondroitin sulfate – by 1.2 times. After transferring the animals to a normal diet, no recovery of the indices was found. According to the studied indicators sodium glutamate has a toxic effect on the liver, pancreas with elements of systemic inflammation. At the stages of discontinuation of monosodium glutamate recovery of most of the studied biochemical markers is not established. Alanine aminotransferase, determined on the 16th day after the abolition of monosodium glutamate, was reduced by 1.33 times compared to the last 8 weeks of admission, gamaglutamyl transpeptidase after 10 weeks was reduced by 1.5 times, after 12 weeks – by 0.4 times, after 14 and 16 weeks, the values corresponded to the control values at 10 week, the alpha-amylase level was reduced by 1.26 times, at 16 weeks – by 2.29 times, but they did not reach the control values. At the end of the study (16 weeks), the level of chondroitin sulfates was increased compared to 8 weeks of feeding animals with glutamate sodium by 1.27 times, and relative to control – by 1.56 times. There was a decrease in glycoprotein content at 16 weeks compared with 8 weeks of observation by 1.3 times, but did not reach the level of intact animals. Conclusion. Thus, the results of serum biochemical studies of rats treated with monosodium glutamate indicated the development of intoxication and its effect on biochemical markers, which were reflected in the dynamics of enzyme activity, inflammation and fibrosis. Most biochemical markers (except gamaglutamyl transpeptidase and aspartate aminotransferase) at the end of the experiment did not reach the level of intact animals, which is apparently due to the chronicity of the pathological process. In rats after discontinuation of monosodium glutamate from 10 to 16 weeks of the experiment, changes in biochemical parameters were observed, which indicated a toxic effect, which was accompanied by the development of subacute inflammatory process. After the transfer of animals to a normal diet, recovery is not established. Sodium glutamate according to the studied parameters has a toxic effect on the liver, pancreas with elements of systemic inflammation

Determining the influence of protein-mineral additives on the properties of butter cookies emulsion

This paper reports the results of studying the influence of two types of protein-mineral additives on the properties of butter biscuit emulsion. The additives are considered as a source of digestible calcium compounds and as a functional and technological component that can improve the quality of buttery flour products. The parameters for pre-hydration of additives in the environment of cow's milk for better implementation of their functional and technological characteristics have been substantiated. It was established that the use of protein-mineral additives in the manufacture of emulsions in the amount of up to 7 % leads to an increase in the emulsification capacity of model systems by 1.5...1.65 times. Improved emulsion resistance has been proven, in particular after heat treatment. It was established that using 5...7 % of the additive produces a pronounced thermal stabilizing effect. After heat treatment at a temperature of 90...95 °C during 3×60 s, when using the protein-mineral additive, a volume of the released water and fat phase increases by 12...25 %. When applying the improved additive, a volume of the released phases increases by 3...10 %. A lower degree of coalescence of the fat phase as part of the emulsion when using the improved protein-mineral additive was microscopically confirmed. The fact of increasing the effective viscosity of emulsions when using up to 7 % of the improved protein-mineral additive was established. This is a positive fact in terms of stabilizing the emulsions and improving their stability as one of the important factors related to the quality of finished flour confectionery. It was established that the improved form of the additive, due to the content of chondroitin sulfates, provides for a better effect on the characteristics of emulsions, which should have a positive influence on the quality of flour-based buttery products.

Dynamics of connective tissue metabolites in the blood of rats of different age under hypokinesia

Topicality. Hypokinesia is a condition of insufficient motor activity of the body with limited pace and volume of movement, and is currently the fourth leading cause of endemic death in the world. It is known that hypokinesia can affect the metabolism of components of bone and cartilage, which is the basis for the development of pathological processes, but there are no biochemical markers of this process in the literature. Thus, currently, there is insufficient number of experimental scientific works devoted to the study of biochemical parameters characterizing the state of the connective tissue in the experiment to assess the impact of hypokinesia on the body, and this fact determines the relevance of the study. Aim. To study the dynamics of the main metabolites characterizing the state of the connective tissue in the blood serum in hypokinesia in rats of different ages. Materials and methods. The studies were performed on 42 white male rats aged 3 and 12 months, kept in the vivarium of the Sytenko Institute of Spine and Joint Pathology National Academy of Medical Sciences of Ukraine, Kharkiv. The conditions of hypokinesia were reproduced using a specially designed device that limited the mobility of the animal without disturbing the ventilation of the body. Animals were removed from the experiment by decapitation in Day 7 and 30 of immobilization under thiopental anesthesia. The content of sialic acids in the serum was determined by the Hess method, chondroitin sulfate – by Nemeth-Csoka method modified by L. I. Slutsky, the fractional composition of glycosaminoglycans (GAG) – by the reaction with resoquine. Results and discussion. In the study of the rat serum, it was found that in intact animals of 3 and 12 months of age the content of sialic acids was different; it was 2.94 ± 0.16 and 1.98 ± 0.09 mmol/L, respectively. After 7 days of hypokinesia, their concentration in young animals did not change, but in older animals, this figure increased. By Day 30, the serum sialic acid levels suddenly increased compared to intact animals. In animals of 12 months of age, the concentration of sialic acids in the blood serum increased by both Day 7 and Day 30 of hypokinesia, especially at the end of the experiment. Hypokinesia also showed a significant increase in serum concentrations of chondroitin sulfates on Day 7 of the experiment in animals of both age groups. At the same time, the level of these metabolites on Day 30 was at the same level. In young rats on Day 7 the fraction of GAG remained unchanged, but on Day 30 there was a significant decrease. During this period, the second fraction of GAG increased. In animals aged 12 months on Day 7 and 30, the content of most GAG fractions was lower than the values characterizing the fractional composition of GAG in intact rats. Conclusions. Thus, the determination of sialic acids, GAG fractions and chondroitin sulfates in the serum can be presented as a set of biochemical tests to assess disorders of the connective tissue metabolism while limiting the motor activity of the body. It has been found that the 30-day hypokinesia causes significant disorders of the connective tissue, and it is reflected in changes in the biochemical parameters of the serum of experimental rats.

Chondroitin 6-sulfate represses keratinocyte proliferation in mouse skin, which is associated with psoriasis

Chondroitin sulfates are implicated in epidermal biology, but functional significance of chondroitin sulfates remains unclear. Here, we report that chondroitin 6-sulfate is important for the maintenance of epidermal homeostasis. Mice deficient in chondroitin 6-O-sulfotransferase-1 (C6st-1), which is involved in biosynthesis of chondroitin 6-sulfate, exhibited keratinocyte hyperproliferation and impaired skin permeability barrier function. Chondroitin 6-sulfate directly interacted with the EGF receptor and negatively controlled ligand-induced EGF receptor signaling. Normal function of hyperproliferative C6st-1-knockout mouse-derived keratinocytes was rescued by treatment with exogenous chondroitin 6-sulfate. Epidermal hyperplasia, induced using imiquimod, was more severe in C6st-1-knockout mice than in C6st-1 wild-type mice. Taken together, these findings indicate that chondroitin 6-sulfate represses keratinocyte proliferation in normal skin, and that the expression level of C6st-1 may be associated with susceptibility to psoriasis.

Deciphering Structural Determinants in Chondroitin Sulfate Binding to FGF-2: Paving the Way to Enhanced Predictability of Their Biological Functions

Controlling chondroitin sulfates (CSs) biological functions to exploit their interesting potential biomedical applications requires a comprehensive understanding of how the specific sulfate distribution along the polysaccharide backbone can impact in their biological activities, a still challenging issue. To this aim, herein, we have applied an “holistic approach” recently developed by us to look globally how a specific sulfate distribution within CS disaccharide epitopes can direct the binding of these polysaccharides to growth factors. To do this, we have analyzed several polysaccharides of marine origin and semi-synthetic polysaccharides, the latter to isolate the structure-activity relationships of their rare, and even unnatural, sulfated disaccharide epitopes. SPR studies revealed that all the tested polysaccharides bind to FGF-2 (with exception of CS-8, CS-12 and CS-13) according to a model in which the CSs first form a weak complex with the protein, which is followed by maturation to tight binding with kD ranging affinities from ~1.31 μM to 130 μM for the first step and from ~3.88 μM to 1.8 nM for the second one. These binding capacities are, interestingly, related with the surface charge of the 3D-structure that is modulated by the particular sulfate distribution within the disaccharide repeating-units.