Acid-soluble Collagen Fractions Research Articles

SUITABILITY OF ACID-SOLUBLE AND ACID-INSOLUBLE LEATHER FRACTIONS IN RADIOCARBON DATING

ABSTRACTLeather has been produced by a variety of methods throughout human history, providing researchers unique insight into multiple facets of social and economic life in the past. Archaeologically recovered leather is often fragile and poorly preserved, leading to the use of various conservation and restoration efforts that may include the application of fats, oils, or waxes. Such additives introduce exogenous carbon to the leather, contaminating the specimen. These contaminants, in addition to those accumulated during interment, must be removed through chemical pretreatment prior to radiocarbon (14C) dating to ensure accurate dating. DirectAMS utilizes organic solvents, acid-base-acid (ABA) and gelatinization for all leather samples. Collagen yield from leather samples is variable due to the method of production and the quality of preservation. However, evaluating the acid-soluble collagen fraction, when available, provides the most accurate 14C dates for leather samples. In instances where gelatinization does not yield sufficient material, the resulting acid-insoluble fraction may be dated. Here we examine the effectiveness of the combined organic solvent and ABA pretreatment with gelatinization for leather samples, as well as the suitability of the acid-insoluble fraction for 14C dating.

New Strategy to Cope with Common Fishery Policy Landing Obligation: Collagen Extraction from Skins and Bones of Undersized Hake (Merluccius merluccius).

In order to promote sustainable fishing practices within European fishing fleets and to avoid the large waste of valuable fish biomass through the practice of fish discarding, the new reform of the common fisheries policy includes the obligation of landing all species under total allowable catch (TAC) regulations. The new policy also prohibits the use of specimens under minimum conservation reference size for direct human consumption. In this context, it is necessary to find new uses for undersized fish, which might help to alleviate the costs associated with the landing obligation but without prompting the creation of a market. European hake (EH) (Merluccius merluccius), which is one of the most important commercial fish species for the Spanish fishing industry, with a total TAC for 2018 of 37,423 t, is used for this study. Consistent with the current policy framework and taking into account the commercial importance of this species, the aim of this work is to study a new strategy for the extraction of collagen from the skin and bone fraction of Merluccius merluccius undersized discards. Three collagen fractions are successfully isolated for the first time from the skin of M. merluccius skin and bone discarded raw material: acid-soluble collagen (ASC) fraction 1 and pepsin-soluble collagen (PSC) fraction 2 from the skin and ASC fraction 3 from bones. The total collagen yield of the process is 13.55 ± 3.18% in a dry basis (g collagen/100 g of skin and bone fraction (SBF)) and 47.80 ± 9.83% (g collagen/100 g of collagen determined by the hydroxyproline content in SBF). The three fractions are further characterized by using different physical and chemical analysis techniques, with the conclusion drawn that the triple helix structure is preserved in the three fractions, although ASC fractions (F1 and F3) present more or stronger hydrogen bonds than the PSC fraction (F2). With the process herein presented, deboned and skinned hake specimens could represent an interesting source of high quality type I collagen, which could be useful as a raw material for the biomedical, cosmetic, and nutraceutical industries.

165 T-Cell Derived Microparticles Induce Fibrolytic Activation of Hepatic Stellate Cells via ICAM-1 (CD54) Dependent Uptake and Emmprin (CD147)

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.

162 Progressive Matrix Crosslinking Limits Reversibility of Liver Fibrosis and is Independent of Tissue Transglutaminase

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.

163 KLF11 Displays Antifibrogenic Effects by Transcriptional Silencing of Most of the Collagen Genes in Hepatic Stellate Cells

Background: Targeting extracellular matrix stability is an attractive antifibrotic strategy. Prior studies have suggested a role for tissue transglutaminase-mediated collagen crosslinking in matrix stabilization, but the extent and mechanisms remain poorly understood. We therefore used a quantitative biochemical approach to characterize dynamic changes in collagen crosslinking during progression and reversal of CCL4-induced liver fibrosis in wildtype mice and in mice deficient in transglutaminase 2 (TG2), the predominant transglutaminase isoform in the liver. Materials and Methods: Liver fibrosis was induced in TG2-/mice and wildtype littermates (C57BL6) by repeated gavage with carbon tetrachloride (CCL4) 3x per week for 1, 3, 6 and 12 weeks. Fibrosis reversal was monitored for up to 36 weeks after CCL4 withdrawal. Collagen crosslinking was measured by sequential extraction using neutral salt, acetic acid and pepsin. Collagen was quantified biochemically via hydroxyproline in the livers, in the extracts and in residual liver tissue. This was correlated to histological evolution and transcript levels of fibrosis related genes. Results: In normal liver, neutral salt, acetic acid and pepsin solubilized 3.2, 1.2% and 91% of total collagen, respectively, while 4.5% remained insoluble, representing a highly cross-linked collagenous matrix. CCL4 administration for 12w led to development of cirrhosis and a 6-fold increase in total liver collagen. Collagen solubility during fibrosis progression revealed progressive cross-linking, with an increase of the insoluble collagen fraction to 27% (6-fold) at week 12 at the expense of the pepsin-soluble fraction. The saltand acid-soluble collagen fractions, representing freshly synthesized collagen, remained constant at 5% of total collagen. The extent of matrix deposition and stabilization remained constant up to 36 weeks after discontinuation of CCL4. Furthermore, no difference in collagen solubility and no fibrosis reversal or change in matrix-related transcripts were found in TG2-/vs. wildtype mice during all time points of progression or follow-up. Conclusions: 1) Quantitative measurement of fibrotic matrix stability revealed a significant increase of collagen crosslinking, which parallels fibrosis progression and limits its reversal. 2) Matrix stabilization in fibrotic liver is independent of transglutaminase-mediated cross-linking since it was unaffected in TG2-/mice 3) This TG2independent collagen cross-linking is an important target for future antifibrotic therapies.

Changes in collagen fractions of the myocardium induced by mechanical unloading of the left ventricle

Improvement of cardiac function by mechanically (assist device) unloading the left ventricle is frequently observed. The composition of different collagen fractions in the extracellular matrix may be a predictor for improvement. We examined the Neutral Salt Soluble Collagen (NSC) and the Acid Soluble Collagen (ASC) content in the myocardium taken from three groups of patients. Group 1 (N 18): healthy myocardium (unused donor hearts). Group 2 (N 8) comprises two subgroups, 2a: myocardium of patients taken at device implantation; 2b: myocardium of the same patients taken at device removal (transplantation). Group 3 (N 10): myocardium taken at device placement from later on weaned patients (no transplantation). The mean amount of NSC of group 1 (3.61 g/mg) did not significantly differ from that of group 2a (3.16 g/mg), P 0.507, however, after unloading it showed a significant increase in group 2b (4.69 g/mg), P 0.2000. At device placement, group 3 showed significant higher amounts of NSC (5.63 g/mg) than in group 1 (P 0.0001) and than in group 2a and 2b (P 0.002; P 0.028). As compared to group 1 (1.77 g/mg), the mean amount of ASC was significantly reduced in group 2a (0.20 g/mg), P 0.0001 and after unloading in group 2b (0.62 g/mg), P 0.001. Groups 1, 3 (1.31 g/mg) and 2b (0.62 g/mg) did not differ statistically (P 0.114, P 0.060), but group 2a showed a statistically lower ASC content than group 3, P 0.002. Ejection fraction (EF) before device placement was below 20% in all patients. At device removal, the EF of group 2 patients (transplanted) was below 35% and group 3 patients (weaned off ) above 50%. Mechanical unloading leads to an increase of NSC and ASC. It leads to NSC increasing above the level of the control myocardium. ASC remains significantly below the control level. The increase in collagen fractions could be regarded as an expression of the reverse of the maladaptive myocardial interstitial remodeling. The weaned patients already showed a hyper-normal NSC and a normal ASC fraction at implantation. This collagen constellation seemed to cope better with the disease than that of the other patients.

Elucidation of the effect of formaldehyde and lipids on frozen stored cod collagen by FT-Raman spectroscopy and differential scanning calorimetry.

The effect of frozen storage (-10 and -30 degrees C), formaldehyde, and fish oil on collagen, isolated from cod muscle, was investigated. Salt- and acid-soluble collagen fractions as well as insoluble collagen indicated changes in solubility on frozen storage. Differential scanning calorimetry (DSC) showed a highly cooperative transition at 28.2 degrees C for isolated collagen. Changes in the thermodynamic properties of collagen were observed on frozen storage at -10 degrees C compared with the control at -30 degrees C because of changes in structure. In the presence of formaldehyde, there were no changes in the DSC collagen transition; however, in the presence of fish oil there was an increase in enthalpy and an extra peak was observed at 44.6 degrees C, indicating collagen-fish oil interaction. Structural changes resulted in a decrease in the solubility of collagen in salt and acid solution. FT-Raman spectra obtained for collagen at -10 degrees C and -30 degrees C confirmed the alteration of the conformation of collagen not only at -10 degrees C but also in the presence of formaldehyde and fish oil.

Effects of dietary xylitol on collagen content and glycosylation in healthy and diabetic rats

The effects of three-month dietary xylitol supplementation on the amounts and hexose contents of acid-soluble collagen as well as on the amounts and fluorescence of collagenase-soluble collagen were studied in healthy and streptozotocin-diabetic male rats. Collagen was extracted from skin samples. In the healthy rats, supplementation with xylitol (10%) increased the hydroxyproline content of the acid-soluble fraction and skin thickness. In diabetic rats receiving and not receiving xylitol, the acid-soluble collagen fraction was markedly lower than in healthy rats. However, its amount was significantly elevated when xylitol had been added to the diet. Supplementation with xylitol caused no changes in the amounts of collagenase-soluble fraction in either healthy or diabetic rats. Supplementation with xylitol (10%) significantly decreased the hexose content of acid-soluble collagen and the fluorescence of the collagenase-soluble fraction in both healthy and diabetic rats. The results indicate that dietary xylitol affects collagen synthesis and collagen glycosylation.

Enzymatic isolation of human trophoblast and culture on various substrates: Comparison of first trimester with term trophoblast

A simple method is described for the isolation of trophoblast cells from both first trimester and term placenta. Trophoblast preparations were characterized by light microscopy, scanning and transmission election miscroscopy and immunohistochemistry to distinguish these cells from mesenchyme and endothelium. Trophoblast cells were cultured on various substrates and a comparison made of their ability to attach, proliferate and function. A collagen gel substrate produced by repolymerization of an acid soluble collagen fraction from chorionic villi allowed rapid attachment of trophoblast cells and maintainance of their original morphology. Term trophoblast cells were shown to become fully functional in short term (three day) cultures by virtue of their increased immunocytochemical staining for the presence of beta hCG, hPL and SPI. beta hCG increased significantly by day three thus demonstrating functional activation. Trophoblast cells from first trimester placenta formed proliferating colonies of hormone producing cells while those from term placenta reaggregated into clusters and closely resembled syncytiotrophoblast both morphologically and functionally. This short term culture system for term trophoblast will allow further studies into the biology of trophoblast polypeptide hormone synthesis and secretion.

Immune-induced dermal connective tissue alterations in rabbits chronically immunized with bovine serum albumin: Biochemical studies on collagen, glycosaminoglycans, RNA, and DNA

Rabbits were chronically immunized by repeated intravenous injections of bovine serum albumin and compared to saline injected controls. All sensitized animals developed antibodies to albumin, but in only a few were circulating immune complexes detected. The dermal biosynthesis of dermatan and heparan sulfate in vivo was significantly restrained following immunization due to a reduced cellular synthesis. The collagen type III/I ratio was significantly repressed in the skin of immunized rabbits, in particular in the acid soluble collagen fraction containing newly formed collagen. Further, the hydroxyproline/DNA ratio, expressing the collagen accumulation per cell, was increased, indicating that the secretion of collagen type I was enhanced. These alterations in the formation and accumulation of matrix components were not correlated to the occurrence of circulating immune complexes or the antibody response. Thus, compared to previous investigations on vascular connective tissue, persistent immunostimulation may alter connective tissue metabolism systemically, and it appears that the metabolism of matrix components characteristic of early connective tissue formation and inflammation is particularly susceptible. These observations may be of significance as regards systemic involvement and chronicity in various inflammatory connective tissue diseases.

Studies on Collagen of Bovine Cornea

Soluble collagen in bovine corneal stroma was determined by homogenizing and sequentially extracting the tissue with 0.45 m NaCl, pH 7.4, 0.5 m citrate buffer, pH 4.3, and 0.5 m acetic acid. Calf cornea contains about three times more soluble collagen than cow cornea. In both calf and cow, the salt-soluble fraction contains the most collagen. Though calf corneas contain citrate and acetic acid-soluble collagen fractions, they are reduced greatly in the cow cornea. Calf corneas were incubated with 3,4–3H-proline for estimation of collagen biosynthesis and the data show that cornea has the capacity for in vitro synthesis of collagen. The tissue utilized radioactive proline slightly better when stroma alone without the epithelium was used. The specific activity of collagen was high in the salt-soluble fraction and also in the dialyzed incubation medium (containing collagen and soluble protein leached from the corneas). With the in vitro system, radioactivity was detected in the insoluble fibrous collagen in the corneas. Chelating agents, antibiotics, and other metabolic inhibitors markedly inhibited biosynthesis of collagen and other proteins. The iron chelator, α, α’-dipyridyl, selectively inhibits biosynthesis of corneal collagen.

Changes in collagen with age—I: The extraction of acid soluble collagens from the skin of mice

Methods of obtaining acid-soluble collagen fractions are compared. Extraction with 0·5M acetic acid at 40°C for 60 min periods is found to be the preferred method for mice skins. The extraction patterns for SM/J mice vary markedly with age.