Low Molecular Weight Degradation Products Research Articles

Fast in vitro hydrolytic degradation of polyester urethane acrylate biomaterials: Structure elucidation, separation and quantification of degradation products

Synthetic biomaterials have evoked extensive interest for applications in the field of health care. Prior to administration to the body a quantitative study is necessary to evaluate their composition. An in vitro method was developed for the quick hydrolytic degradation of poly-2-hydroxyethyl methacrylate (pHEMA), poly(lactide-co-glycolide50/50)1550-diol (PLGA(50:50)1550-diol), PLGA(50:50)1550-diol(HEMA)2 and PLGA(50:50)1550-diol(etLDI-HEMA)2 containing ethyl ester lysine diisocyanate (etLDI) linkers using a microwave instrument. Hydrolysis time and temperature were optimized while monitoring the degree of hydrolysis by 1H NMR spectroscopy. Complete hydrolytic degradation was achieved at 120°C and 3bar pressure after 24h. Chemical structure elucidations of the degradation products were carried out using 1H and 13C NMR spectroscopy. The molecular weight (MW) of the polymethacrylic backbone was estimated via size-exclusion chromatography coupled to refractive index detection (SEC-dRI). A bimodal MW distribution was found experimentally, also in the pHEMA starting material. The number average molecular weights (Mn) of the PLGA-links (PLGA(50:50)1550-diol) were calculated by high pressure liquid chromatography–time-of-flight mass spectrometry (HPLC–TOF-MS) and 1H NMR. The amounts of the high and low MW degradation products were determined by SEC-dRI and, HPLC–TOF-MS, respectively. The main hydrolysis products poly (methacrylic acid) (PMAA), ethylene glycol (EG), diethylene glycol (DEG), lactic acid (LA), glycolic acid (GA) and lysine were recovered almost quantitatively. The current method leads to the complete hydrolytic degradation of these materials and will be helpful to study the degradation behavior of these novel cross-linked polymeric biomaterials.

Complex Flavonoids in Cocoa: Synthesis and Degradation by Intestinal Microbiota

Rarely occurring flavan-3-ol derivatives such as C-glycosides can be generated during food processing, for example, by cocoa production. These astringent taste compounds may also exert interesting behavior toward microbial metabolism, as other C-glycosides have been shown to be quite stable. Oligomeric flavan-3-ols, the procyanidins, bear also a C-C bond between the main moieties and are suspected to resist microbial metabolism for a prolonged time compared to other flavonoids. This paper describes a semisynthetic approach for the generation of flavan-3-ol C-glycosides. Results of incubation experiments studying five flavan-3-ol C-glycosides bearing different sugars, linkage positions, and stereochemistries are presented as well as the behavior of di- and trimeric B-type procyanidins toward intestinal microbiota. Low molecular weight degradation products are considered as well as concentration-time courses of degraded and liberated compounds. All metabolic studies were performed with the well-proven pig cecum model.

Structural characterization of residual lignins isolated with cyanamide‐activated hydrogen peroxide from various organosolvs pretreated wheat straw

AbstractThe posttreatment of various organosolvs pretreated wheat straw with cyanamide‐activated hydrogen peroxide was studied. About 44–80% of the total residual lignin and 38–85% of the total residual hemicelluloses were released or degraded during the posttreatment with 1.8% H2O2–0.18% cyanamide at 50°C under pH 10.0 for 4 h from different aqueous organic acids or alcohols pretreated straw. The seven degraded residual lignin preparations were subjected to a comprehensive physicochemical and structural characterization by UV, FTIR, and 1H and 13NMR spectroscopy, and GPC. The nitrobenzene oxidation method was also applied to the in situ lignins. It was found that the seven residual lignin preparations contained large amounts of noncondensed syringyl and guaiacyl units, together with fewer noncondensed p‐hydroxyphenyl units, esterified p‐coumaric acid, and mainly etherified ferulic acid. All of the lignin fractions are free of associated polysaccharides and had molecular‐average weights ranging between 2980 and 3820 g mol−1. Analysis of these low molecular weight degradation products revealed an oxidation of residual lignin had occurred. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008

MALDI−TOF MS and DIOS−MS Investigation of the Degradation and Discoloration of Poly(ethylene terephthalate)

In this study, three mass spectrometry techniques matrix-assisted laser desorption ionization mass spectrometry (MALDI−TOF MS) combined with thin-layer chromatography (TLC), laser desorption/ionization on silicon−mass spectrometry (DIOS−MS), and time-of-flight secondary ion mass spectrometry (TOF−SIMS) were used to investigate the thermooxidative degradation and resultant discoloration of poly(ethylene terephthalate) PET that frequently occurs during melt processing. The MALDI−TOF MS spectra of the degraded polymer showed the formation of oligomers containing carboxyl end groups via chain scission at the ether link present in PET. In addition, a variety of cyclic oligomers are found to form via two different mechanisms from the linear oligomers of the virgin PET. Thus, our results not only confirm the oligomers containing carboxyl end groups and cyclic oligomers as the low molecular weight degradation products but also deepen the understanding about their structures, molecular weights, and molecular weight distributions. Furthermore, a MALDI−TOF MS study directly on the TLC plate and DIOS−MS enable us to identify low molecular weight compounds (not detected previously due to matrix interference) that are contributors to color formation.

Solid-phase microextraction for qualitative and quantitative determination of migrated degradation products of antioxidants in an organic aqueous solution

Low molecular weight aromatic substances may migrate out from plastic packaging to their contents, especially if they consist of organic aqueous solutions or oils. It is, therefore, extremely important to be able to identify and quantify any migrated substances in such solutions, even at very low concentrations. We have in this work investigated and evaluated the use of solid-phase microextraction for the specific task of extraction from an organic aqueous solution such as a simulated pharmaceutical solution consisting of 10 vol.% ethanol in water. The goal was furthermore to investigate the possibility of simultaneously identifying and quantifying the substances in spite of differences in their chemical structures. Methods were developed and evaluated for extraction both with direct sampling and with headspace sampling. Difficulties appeared due to the ethanol in the solution and the minute amounts of substances present. We have shown that a simultaneous quantification of migrated low molecular weight degradation products of antioxidants using only one fibre is possible if the extraction method and temperature are adjusted in relation to the concentration levels of the analytes. Comparions were made with solid-phase extraction.

Low‐Molecular Weight Advanced Glycation End Products: Markers of Tissue AGE Accumulation and More?

Incomplete digestion of advanced glycation end product (AGE)-modified protein results in the formation of low-molecular weight degradation products incorporating AGE modifications (LMW-AGEs). In addition to being biomarkers of AGE modification, LMW-AGEs may have a high toxic potential, being free to interact with AGE receptors at distant sites via the circulation. Several free AGEs have been identified, including pentosidine, N(epsilon)-(carboxymethyl)lysine (CML), and free-imidazole AGEs. In addition, fluorescence (370 nm [excitation]/440 nm [emission]) in the LMW phase of serum correlates with tissue fluorescence, an established marker for AGE modification. In experimental diabetes, LMW fluorescence increases with duration of disease and is normalized with the AGE inhibitor aminoguanidine. LMW fluorescence is also higher in patients with diabetes, in whom it is associated with glomerular filtration rate and hemoglobin. Patients with hyperfiltration have lower LMW fluorescence than those with normal renal function, which may protect them from AGE accumulation in the short term. These findings provide clinical support for the association between AGEs and progressive renal injury in diabetes.

Improvement of the Wetting and Absorption Properties of Lignocellulosic Fibers by Means of Gas Phase Ozonation

Gas phase ozonation was done on sheets made from chemical thermomechanical pulp in order to improve the wetting properties of the lignocellulosic fibers. The degree of modification was varied by letting the reaction continue for different lengths of time, ranging from 1 to 60 min. Changes in the chemistry of the fibers after ozone exposure were investigated using Fourier transform infrared (FT-IR) spectroscopy and electron spectroscopy for chemical analysis (ESCA). The evolution of a carbonyl signal and the decrease of aromatic absorption over time was observed with FT-IR spectroscopy. The carbonyl peak grew in intensity as the reaction continued throughout the whole range of treatment times. The ESCA showed that carbonyl and carboxyl functionalities were introduced after 10 min of ozone exposure and that the intensity of the peak from the aliphatic and aromatic carbons decreased. However, an ozone treatment longer than 15 min did not affect the chemical surface composition, as analyzed by ESCA. The single-fiber contact angle with water, measured using a Cahn balance, decreased with extended ozonation. Measuring the time required for the sheet to absorb a water droplet with a high speed camera showed that even a very short ozone exposure (1 min) dramatically affected the absorption behavior. The rate of absorption dramatically increased after as little as 1 min of ozone exposure. This improvement in absorption rate was most likely due to the formation of low molecular weight degradation products, acting as wetting agents, created during the ozonation.

Formation and chemical structures of acid-soluble lignin II: reaction of aromatic nuclei model compounds with xylan in the presence of a counterpart for condensation, and behavior of lignin model compounds with guaiacyl and syringyl nuclei in 72% sulfuric acid

To elucidate the formation and chemical structures of water-soluble material in acid-soluble lignin (ASL), lignin aromatic nuclei model compounds of creosol (I) and 5-methoxycreosol (II) were reacted with xylose or xylan in the presence of apocynol as a counterpart for condensation in 72% sulfuric acid (SA). The reaction of I gave mainly condensation product. However, the condensation reaction of II with apocynol was suppressed because of steric hindrance from the methoxyl group, and II yielded a C-xyloside after refluxing in 3% SA together with condensation products. To obtain information on CHCl3-soluble material in ASL, model compounds of arylglycerol-β-aryl ethers with guaiacyl (VIII) and syringyl (X) nuclei were treated by the Klason procedure. VIII gave only insoluble polymerized product, while X gave insoluble polymerized product and CHCl3-soluble low molecular weight products, which were dissolved in 3% SA. These results prove earlier views that water-soluble material in ASL consists of condensation products formed from syringyl lignin and monosaccharide units in hemicellulose. In addition, the CHCl3-soluble material in ASL appears to be composed of low molecular weight degradation products from SA treatment of Klason lignin with the syringyl nucleus.

Differential effect of transforming growth factor beta (TGF-beta) on the genes encoding hyaluronan synthases and utilization of the p38 MAPK pathway in TGF-beta-induced hyaluronan synthase 1 activation.

Unfettered hyaluronan (HA) production is a hallmark of rheumatoid arthritis. The discovery of three genes encoding hyaluronan synthases (HASs) allows for the investigation of the signaling pathways leading to the activation of these genes. Our objective is to further understanding of the regulation of these genes as well as to find ways to prevent undesired gene activation. Human fibroblast-like synoviocytes were used in these experiments. mRNA levels of HAS were monitored by reverse transcriptase-PCR. A series of specific kinase inhibitors were used to investigate intracellular pathways leading to the up-regulation of HAS1. Our experiments, testing a series of stimuli including tumor necrosis factor alpha (TNFalpha), demonstrate that TGF-beta is the most potent stimulus for HAS1 transcription. TGF-beta activates HAS1 in a dose-dependent manner with a maximum effect at a concentration of 0.5-1 ng/ml. TGF-beta-induced HAS1 mRNA can be detected within 60 min and reaches maximal levels at 6 h. Furthermore, TGF-beta treatment leads to an increase in synthase activity as determined by HA ELISA and by in vitro HA synthase assays. In contrast to the activatory effect on HAS1, TGF-beta dose-dependently suppresses HAS3 mRNA. As to the mode of action of TGF-beta-induced HAS1 mRNA activation, our experiments reveal that blocking p38 MAPK inhibited the TGF-beta effect by 90%, blocking the MEK pathway led to an inhibition by 40%, and blocking the JNK pathway had no effect. The presented data might contribute to a better understanding of the role of TGF-beta and of HA in the pathology of diseases.

The influence of temperature and the kind of the atmosphere on polystyrene thermal degradation

The influences of atmosphere and sample heating rate on the degradation temperature were estimated. Thermal degradation of polystyrene (PS) was carried out in a flow tubular furnace under isothermal conditions and in a thermogravimetric analyser under dynamic conditions. The experiments were performed in both a nitrogen and an air atmosphere. The thermal behaviour of tested polymer was determined on the basis of the thermogravimetric curves. The process of polystyrene thermal degradation in the tubular furnace was studied at the temperatures of 300°C, 400°C, 500°C, and 600°C for 30 min. The volatile and less-volatile thermal degradation products were analyzed using gas chromatography - mass spectrometry (GC-MS) and Fourier transform infrared spectroscopy (FT-IR) methods. 64 degradation products were identified. Main of them were styrene and its dimer and trimer. In air atmosphere, the concentration of low molecular weight degradation products increases with the temperature while in nitrogen the maximal concentrations of some of them are reached at temp. 500°C.

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model

To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.

Reactions of Lignin with Cyanamide Activated Hydrogen Peroxide. Part 3. The Degradation of Pine Kraft Lignin

Summary The oxidation of pine kraft lignin by alkaline hydrogen peroxide and cyanamide activated hydrogen peroxide was studied. Over 70% of the kraft lignin was degraded and solublized in the presence of cyanamide as compared to less than 20% by hydrogen peroxide alone. A large decrease in the phenolic hydroxyl and methoxyl content,as well as an increase in the carboxylic acid content was also observed in the insoluble residue. Analyses of the low molecular weight degradation products revealed extensive oxidation of the kraft lignin had occurred,consistent with the ring-opening reactions observed in our previous studies using lignin model compounds.

Thermal oxidation of poly(ethylene oxide–propylene oxide–ethylene oxide) triblock copolymer: focus on low molecular weight degradation products

The oxidative thermal degradation of poloxamer 407, a poly(ethylene oxide–propylene oxide–ethylene oxide) triblock copolymer, at 50 and 80 °C in air was studied by solid phase microextraction/gas chromatography–mass spectrometry (SPME/GC–MS). At 80 °C, we found that degradation was initiated on the PPO block of the copolymer by three mechanisms involving hydroperoxyl formation and depropagation. 1,2-propanediol,1-acetate; 1,2-propanediol,2-formate; 1,2-propanediol,1-acetate,2-formate and 2-propanone,1-hydroxy were the first degradation products produced. Random chain scissions and a sharp decrease in the molecular weight of the material followed the initiation period. Formic acid and acetic acid, formed upon degradation, participated in esterification reactions leading to the formation of the formate and acetate forms of 1,2-propanediol and ethanediol. Though degradation at 50 °C was much slower, the oxidative mechanisms leading to low molecular weight formates and acetates were the same as those observed at 80 °C.

Differences in the modulating potential of advanced glycation end product (AGE) peptides versus AGE proteins

Differences in the modulating potential of advanced glycation end product (AGE) peptides versus AGE proteins. Advanced glycation end products (AGEs), identified as irreversible products of a complex reaction of carbonyl groups of reducing sugars with free protein amino groups, are characterized by resistance to proteolytic degradation. The incomplete digestion of AGEs results in low molecular weight AGEs accumulating in the blood of diabetic and uremic patients. We hypothesized that the accumulation of these compounds may contribute to the dysfunction and/or degeneration of tubular epithelial cells. Our study examined whether low-molecular-weight AGE peptides and high-molecular-weight AGE proteins affect the functional cellular properties of two tubular epithelial cell lines: immortalized human kidney tubular epithelial (IHKE) and immortalized rat renal proximal tubular cells (IRPTCs). Parameters of cellular damage and growth behavior were cell counting, analysis of the cellular metabolic activity (MTT assay), as well as cellular proliferation (3[H]-thymidine-incorporation). IHKE treated with bovine serum albumin-AGE (BSA-AGE 50) or BSA-AGE-Pep 50 revealed a decrease in cellular metabolic activity as compared with controls after 48 hours of incubation (73 +/- 9% for BSA-AGE 50 and 62 +/- 11% for BSA-AGE-Pep 50 vs. 89 +/- 8% for BSA Co 50). Low molecular weight BSA-AGE-Pep 50 induced a significantly greater cellular damage in IRPTCs as compared with high molecular weight BSA-AGE 50 after 144 hours of incubation (59 +/- 15% for BSA-AGE 50 vs. 31 +/- 13% for BSA-AGE-Pep 50). The decrease in metabolic activity correlated well with a decrease in cellular proliferation. The results suggest a higher toxic potential of low molecular weight AGE peptides compared with high molecular weight AGE proteins in IRPTC and IHKE. This may provide evidence that low molecular weight degradation products of AGE-modified proteins have an important risk potential.

Defective Intracellular Processing of Lactase‐Phlorizin Hydrolase Protein in Rats Prenatally Exposed to Ethanol

We have previously shown that fetal exposure to ethanol in rats produces both structural and biochemical abnormalities in absorptive enterocytes. Among the indicators of injury are derangements in the expression of lactase-phlorizin hydrolase (LPH), which is an essential enzyme for the assimilation of milk. In an animal model of fetal alcohol syndrome, unsuckled newborn rats prenatally exposed to maternal ethanol revealed a 10- to 15-fold increase in the number of LPH mRNA molecules per absorptive enterocyte, compared with controls (Estrada et al., Alcohol. Clin. Exp. Res. 20:1662-1668, 1996). However, lactase activity per cell was similar in both groups. The aim of this study was to characterize the effect of prenatal exposure to ethanol on the processing of LPH mRNA and protein. RNase protection assays using 3'- and 5'-directed antisense RNA probes revealed that the LPH mRNA from ethanol-exposed pups is full length. However, metabolic labeling, followed by immunoprecipitation using an anti-LPH monoclonal antibody, demonstrated a significant alteration in LPH protein processing. Intestinal explants from 21-day ethanol-exposed fetuses that were chased 30 min after a [35S]methionine pulse showed greater amounts of newly synthesized LPH precursors (205 and 220 kDa) and low molecular weight degradation products than controls. However, despite the increases in LPH precursor, the amount of 130 kDa mature LPH was similar in ethanol-exposed and control explants. These data suggest an increase in intracellular degradation of LPH precursor in rats prenatally exposed to ethanol, which occurs before its insertion into the microvillus membrane. Biosynthesis of LPH appears to be upregulated at the transcriptional level, which overcomes the degradation of LPH precursor during processing.

Biological basis of enzyme‐catalyzed polyester degradation: 59 C‐terminal amino acids of poly(3‐hydroxybutyrate) (PHB) depolymerase a from pseudomonas lemoignei are sufficient for PHB binding

AbstractBiodegradable polyesters such as biologically produced poly[(R)‐3‐hydroxybutyric acid], (PHB) other polyhydroxyalkanoic acids and related chemosynthetic polyesters have attracted industrial interest, and bacterial produced PHB is commercially available since 1990. A large variety of polyester degrading microorganisms have been found to be present in environment. The microorganisms decompose the polymers by secretion of extracellular polyester depolymerases and utilize low molecular weight degradation products for growth. Microbial polyester depolymerases have the unique property to be water soluble and to be able to bind specifically to polyester surfaces. The objective of this contribution is a functional analysis of a bacterial PHB depolymerase polyester binding domain. In addition, a detailed summary of the present knowledge on the biochemistry of enzymatic polyester hydrolysis is provided.

Microbial degradation of polyesters: a review on extracellular poly(hydroxyalkanoic acid) depolymerases

Thermoplastic polyesters such as biologically produced poly[( R)-3-hydroxy-butyric acid], other polyhydroxyalkanoic acids and related chemosynthetic polyesters, have properties which make them valuable for many applications. In addition, these materials can be biodegraded to water and carbon dioxide. Many aerobic and anaerobic polyester-degrading microorganisms have been found in all moderate ecosystems. The microorganisms decompose the polymers by secretion of extracellular polyester depolymerases and utilize low molecular weight degradation products. This review summarizes the present knowledge on microbial extracellular polyester depolymerases.

Depolymerization processes in the thermal degradation of cellulosic paper insulation in electrical transformers

A dynamic heating experiment was used to examine the influence of temperature on the degradation of the Kraft insulating paper in transformer oil. The degradation was monitored by measuring a number of key indicators on selected samples as a function of progressive ageing. These indicators included the degree of polymerization (DP), level of furanic compounds and level of gases such as carbon oxides and oxygen. The results show that the formation of furanic compounds and of gases begins significantly after a large reduction in DP, indicatinga two-stage process: firstly scission leading to a sharp drop in DP and secondly formation of low molecular weight degradation products.

Molecular characterization of plasminogen activators in human gingival crevicular fluid

Plasminogen activators (PAs), a family of serine proteases, and their inhibitors (PAIs) are important in fibrinolysis, wound healing and tissue remodelling. Previous studies revealed differences in the localization of PA activity between healthy and diseased gingival tissues, suggesting that PAs and PAIs could play a part in periodontal homeostasis and disease. PAs and PAIs are synthesized by most of the cell types making up the periodontium and can be identified in gingival crevicular fluid (GCF). These studies sought to characterize the molecular species of PAs and their inhibitors in GCF collected from clinically healthy sites. PA enzymatic activity in GCF samples demonstrated by fibrin zymography revealed the presence of only tissue-type PA (tPA) activity. No urokinase-type PA (uPA) enzymatic activity was detected. tPA enzymatic activity appeared predominantly as an uncomplexed 70-kDa species, although some samples contained enzyme-inhibitor complexes. Quantitation of total tPA by enzyme immunoassay showed a mean concentration of 1.6ng/μl. Analysis of GCF samples for uPA by immunoblotting and enzyme immunoassay disclosed the presence of small amounts of uPA (0.2 ng/μl), which were present predominantly in activator-inhibitor complexes. Immunoblotting showed specific PAI-2 immunoreactivity bands in high molecular-weight complexes and low molecular-weight degradation products, but less than nanogram amounts of free PAI-2 molecules. Enzyme immunoassay revealed that PAI-2 was present in an at least a seven times greater amount than PAI-1. These observations support the hypothesis that PA-generated proteolysis and its regulation by endogenous inhibitors has a role in the diverse biochemical mechanisms underlying periodontal physiology and pathology including host-microbial interaction, polymorphonuclear leucocyte infiltration, turnover and migration of epithelial cells, connective tissue degradation and remodelling, fibrinolysis and wound healing.

Microbial exopolymers provide a mechanism for bioaccumulation of contaminants

Scanning confocal laser microscopy was used to directly visualize accumulation of the herbicide diclofop methyl and its breakdown products by a degradative biofilm community, cultivated in continuous-flow cell cultures. Some bacterial cells accumulated these compounds. However, most accumulation occurred in cell capsules and certain regions of the exopolymer matrix. Mass spectroscopic analysis of the biofilm material confirmed accumulation of the parent compound and its breakdown products in the biofilms. Lower molecular weight degradation products were found in the effluent, indicating mineralization of diclofop by the flow cell cultures. Grazing protozoa feeding on the biofilms nonselectively ingested cell capsules and exopolymers, suggesting direct transfer and accumulation of the contaminants in protozoa. These findings demonstrated that microbial exopolymers can play an important role in the bioaccumulation of contaminants in natural systems.