Nutritive Fibers of Animal Origin: Collagen and Its Fractions as Essential Components of New and Useful Food Products

Data on the production of collagen and its fractions (which in many cases surpass the parent protein in functional properties) reported in the literature throughout the past two decades are reviewed. The material presented elucidates the role played by collagen and its fractions in the development of new, ecologically pure foodstuffs containing nutritive fibers. As follows from the analysis of these data, collagen fractions with molecular weights in excess of 120 kDa offer the greatest promise for producing useful foodstuffs.     

Effect of cadmium on collagen content and solubility in rat bone

The toxic action of cadmium in the bone tissue is known, but its mechanisms are still unexplained. We examined whether Cd influences collagen content and its solubility in the femoral bone of three-week-old female rats exposed to 5 or 50 mg Cd/l in drinking water. Non-cross linked collagen was extracted with 0.5 M acetic acid, and two acid-insoluble collagen fractions were extracted with pepsin and 4.0 M guanidine hydrochloride, respectively. SDS/PAGE showed the presence of two collagen types, I and V, in all three extracted fractions. Exposure of rats to Cd for 6 months increased the amount of acid-soluble collagens type I and V and decreased the level of acid-insoluble collagens. The amount of total collagen extracted from the bones of rats exposed to 50 mg Cd/l was reduced by about 14% as compared to control and those intoxicated with 5 mg Cd/l. The solubility of type I bone collagen (determined as the percentage of acetic-soluble fraction of total collagen) was increased 2.9- and 3.0-fold in rats intoxicated with 5 and 50 mg Cd/l, respectively. Similarly, the solubility of type V collagen was increased 2.3- and 2.7-fold, respectively. Our results indicate that Cd treatment affects bone collagen by decreasing its content and increasing its solubility.     

Chemical changes associated with aging of collagen in vivo and in vitro

Collagen extracted from rat skin by neutral-salt solutions contains less aldehydes than the more insoluble collagen fractions. The concentration of aldehydes in collagen is directly related to its capacity to form stable cross-linked gels, which do not redissolve on cooling and become more insoluble in a variety of reagents. Whereas the absorption spectrum of neutral-salt-soluble collagen treated with N-methylbenzothiazolone hydrazone resembles that of acetaldehyde, the more insoluble collagen fractions show increasing amounts of a component that behaves like an alphabeta-unsaturated aldehyde. The ratio between alpha- and beta-sub-units present in a particular fraction of soluble collagen seems to be constant and independent of the age of the animal. Neutral-salt-soluble collagen, which has a low concentration of beta-components, will generate intramolecular bonds if gelled at 37 degrees . These intramolecular bonds seem to precede the formation of stable intermolecular cross-links, since these gels can redissolve when cooled to yield a soluble collagen with a higher content of beta-components of intramolecular origin.     

Bone matrix studies Influences of parathyroid extract,calcitonin, and cholecalciferol and of rickets and its treatment

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by paratyroid extract.Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamine D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Bone matrix studies. Influences of parathyroid extract, calcitonin, and cholecalciferol and of rickets and its treatment.

Bones from young rats were incubated with radioactive glucosamine and proline. The concentrations and specific activities of matrix glycosaminoglycan fractions, prepared by a cetylpyridinium chloride method, and the specific activity of insoluble collagen hydroxyproline were determined. Acute parathyroid extract treatment increased labelling of hyaluronic acid and a glycopeptide fraction. These effects were partially blocked by calcitonin treatment which had no effect by itself. Parathyroid extract inhibited collagen synthesis and this effect was not blocked by calcitonin. Effects of these two hormones on labelling of chondroitin sulfate fractions were more variable. Vitamin D-3 caused an increase in labelling of all matrix fractions measured in bone from thyroparathyroidectomized rats, but its stimulating effect upon collagen synthesis was blocked by parathyroid extract. Bones from rats made rachitic on a phosphorus and vitamin D-deficient diet were incubated in vitro with radioactive glucosamine and proline. Over a three-week period rachitic bone exhibited a progressive fall in concentration and labelling of a glycopeptide-hyaluronic acid fraction, while pair-fed animals supplemented either with phosphorus alone or with phosphorus and vitamin D-3 not only remineralized their bones, but the bones showed a pronounced increment in concentration and labelling of this fraction. Both treatment regimens also enhanced chondroitin sulfate and collagen labelling.     

Studies on the glycosylation of hydroxylysine residues during collagen biosynthesis and the subcellular localization of collagen galactosyltransferase and collagen glucosyltransferase in tendon and cartilage cells

1. The glycosylation of hydroxylysine during the biosynthesis of procollagen by embryonic chick tendon and cartilage cells was examined. When free and membrane-bound ribosomes isolated from cells labelled for 4min with [(14)C]lysine were assayed for hydroxy[(14)C]lysine and hydroxy[(14)C]lysine glycosides, it was found that hydroxylation took place only on membrane-bound ribosomes and that some synthesis of galactosylhydroxy[(14)C]lysine and glucosylgalactosylhydroxy[(14)C]lysine had occurred on the nascent peptides. 2. Assays of subcellular fractions isolated from tendon and cartilage cells labelled for 2h with [(14)C]lysine demonstrated that the glycosylation of procollagen polypeptides began in the rough endoplasmic reticulum. (14)C-labelled polypeptides present in the smooth endoplasmic reticulum and Golgi fractions were glycosylated to extents almost identical with the respective secreted procollagens. 3. Assays specific for collagen galactosyltransferase and collagen glucosyltransferase are described, using as substrate chemically treated bovine anterior-lens-capsule collagen. 4. When homogenates were assayed for the collagen glycosyltransferase activities, addition of Triton X-100 (0.01%, w/v) was found to stimulate enzyme activities by up to 45%, suggesting that the enzymes were probably membrane-bound. 5. Assays of subcellular fractions obtained by differential centrifugation for collagen galactosyltransferase activity indicated the specific activity to be highest in the microsomal fractions. Similar results were obtained for collagen glucosyltransferase activity. 6. When submicrosomal fractions obtained by discontinuous-sucrose-density-gradient-centrifugation procedures were assayed for these enzymic activities, the collagen galactosyltransferase was found to be distributed in the approximate ratio 7:3 between rough and smooth endoplasmic reticulum of both cell types. Similar determinations of collagen glucosyltransferase indicated a distribution in the approximate ratio 3:2 between rough and smooth microsomal fractions. 7. Assays of subcellular fractions for the plasma-membrane marker 5'-nucleotidase revealed a distribution markedly different from the distributions obtained for the collagen glycosyltransferase. 8. The studies described here demonstrate that glycosylation occurs early in the intracellular processing of procollagen polypeptides rather than at the plasma membrane, as was previously suggested.     

Influence of thyroid hormones on collagen content in tissues of guinea pigs.

Collagen fractions content and level of collagen catabolites in body fluids were determined in normal, hypo- and hyperthyroid guinea pigs. An increase of urinary excretion of hydroxyproline and hydroxylysine as well as concentration of these amino acids in blood serum was found in hyperthyroidism, and a decrease was shown in hyperthyroid guinea pigs. Hyperthyroidism stimulated an increase of neutral-salt-soluble and acid-soluble collagen in skin and liver, and a decrease of insoluble collagen in skin as well as increase of all collagen fractions in bone samples. Hypothyroidism induced a decrease of all collagen fractions in skin and liver, and an increase of acid-soluble and insoluble collagen in bone samples.     

Isolation and chemical characterization of collagen in bovine pulmonary tissues.

1. The contents of the fibrous proteins collagen and elastin in the pleural and parenchymal regions of bovine lungs were determined. The collagen content was approx. 70% (g/100g of salt-extracted defatted powder) in each tissue, and the elastin content was 28% in pleura and 13.5% in parenchyma. 2. Purification of the insoluble collagen from the pleura and parenchyma of bovine lungs by various methods was attempted. The collagen fractions isolated after incubation of the pulmonary tissues with the proteolytic enzymes collagenase ("collagenase-soluble" fraction) or pancreatic elastase ("elastase-insoluble" fraction) each contained approx. 87% of the total collagen initially present. 3. Both collagen fractions were chemically analysed for their amino acid and carbohydrate contents and were found to be similar to those of the intact interstitial collagens isolated from skin, bone and tendon. 4. The contents of the two aldimine cross-linking compounds, dehydrohydroxylysinonorleucine and dehydrodihydroxylysinonorleucine, were determined in the bovine pulmonary collagen fractions, and were found to decrease with increasing age of the animals, and were similar to the values found in intact collagens from bone and tendon.     

Proteins in the fossil bone of the dinosaur, seismosaurus

Proteins have been successfully extracted from the fossil vertebra of a 150-million-year-old sauropod dinosaur (Seismosaurus) recently excavated from the Morrison Formation of New Mexico. HCl and guanidine·HCl extracts of the fossil bone and its sandstone matrix were concentrated, demineralized, and resolved into a number of different protein fractions by reversed-phase high-performance liquid chromatography (HPLC). One of these fractions had the same retention time as collagen. Amino acid analysis (Pico-Tag method) of these fractions confirmed they were proteins. Comparison of the correlation coefficients of the amino acid analyses with that of collagen standards indicated that none of the fractions contained significant amounts of collagen. Similar HPLC profiles were obtained for the HCl extracts of fossil bone and its sandstone matrix suggesting they contained the same proteins. However, different HPLC profiles were obtained when these HCl extracts were dried and reextracted with guanidine·HCl. These different fractions represent proteins unique to the fossil and were not found in the sandstone matrix. These differences were confirmed by amino acid analysis. Such information on fossil bone proteins might provide useful knowledge concerning the evolution of skeletal molecules and the fossilization process. Similar information on the proteins from the geological matrix might provide useful fingerprints for reconstructing ancient environments and for assessing sedimentary rocks for fossil fuel exploration.     

Changing cultures, changing cuisines: Cultural transitions and dietary change in Iron Age, Roman, and Early Medieval Croatia

Food is well-known to encode social and cultural values, for example different social groups use different consumption patterns to act as social boundaries. When societies and cultures change, whether through drift, through population replacement or other factors, diet may also alter despite unchanging resource availability within a region. This study investigates the extent to which dietary change coincides with cultural change, to understand the effects of large-scale migrations on the populations' diets. Through stable carbon and nitrogen isotope analysis of Iron Age, Roman, and Early Medieval human bone collagen, we show that in Croatia large-scale cultural change led to significant changes in diet. The isotopic evidence indicates that Iron Age diet consisted of C(3) foodstuffs with no isotopic evidence for the consumption of C(4) or marine resources. With the Roman conquest, marine resources were added to the diet, although C(3) foodstuffs continued to play an important role. In the Early Medieval period, this marine component was lost and varying amounts of C(4) foodstuffs, probably millet, were added to the otherwise C(3) diet. In both of these transitions it is likely that the changes in diet are related to the arrival of a new people into the area.     

Metalloproteinases from rabbit bone culture medium degrade types IV and V collagens, laminin and fibronectin.

Gel-filtration chromatography of culture medium from rabbit bone explants separates three latent metalloproteinases with activities against collagen, proteoglycan and gelatin respectively. The fractions degrading proteoglycan also degrade laminin, fibronectin and the polymeric products of pepsin-solubilized type IV collagen and can also solubilize insoluble type IV collagen. The fractions degrading gelatin are capable of degrading solubilized type V and 1 alpha,2 alpha,3 alpha (cartilage) collagens, as well as the lower-molecular-weight products of pepsin-solubilized type IV collagen. All activities can be inhibited by 1,10-phenanthroline and occur in either partially or totally latent forms that can be activated by 4-aminophenylmercuric acetate.     

The relationship between reversibility of fibril formation and subunit composition of rat skin collagen

1. Salt-soluble rat skin collagen was precipitated from solution at neutral pH and 37 degrees . On cooling, a portion of the collagen returned into solution. The fractions were separated, the supernatant was concentrated and the precipitate was redissolved in dilute acetic acid. 2. Solutions of supernatant and precipitate were subjected to the same fractionation procedure, giving four fractions. 3. Each fraction was examined by starch-gel electrophoresis and a relationship between subunit composition and the fractionation procedure was noted. The collagen that redissolved on cooling contained less of the more highly cross-linked components than did either the fraction remaining in the precipitate or the starting material.     

Further biochemical and physicochemical characterization of minor disulfide-bonded (type IX) collagen, extracted from foetal calf cartilage

Minor disulfide-bonded collagen (previously termed X1-X7 and now called type IX collagen) was isolated from foetal calf cartilage after pepsin treatment. At least three native fractions, containing, respectively, the X1X2X3, X4, and X5X6X7 chains, were separated; and from further biochemical and physicochemical experiments (differential scanning calorimetry, electrical birefringence, rotary shadowing), we propose a tentative model for their organization within a parent molecule. X1 and X2 are molecules composed of three chains of apparent Mr 62,000 and 50,000 linked by interchain disulfide bonds and containing pepsin-sensitive regions. The cleavage of at least three of these sites, present within X2, gives rise to the X3 and X5X6X7 fractions composed of molecules 80-100 nm and 40-55 nm in length, respectively. The X5X6X7 fraction is not digested by pepsin at 30 degrees C owing to its high thermal stability (certainly explained by its high hydroxyproline + proline content). This organization is in good accordance with that proposed for chicken cartilage type IX collagen; differences could only exist in the number and (or) the location of the pepsin-sensitive sites.     

The molecular stoichiometric hydration model (SHM) as applied to tendon/collagen, globular proteins and cells

This report describes and documents the presence of multiple water-of-hydration fractions on proteins and in cells. Initial studies of hydration fractions in g of water/g of DM (dry mass) for tendon/collagen led to the development of the molecular SHM (stoichiometric hydration model) and the development of methods for calculating the size of hydration fractions on a number of different proteins of known amino acid composition. The water fractions have differences in molecular motion and other physical properties due to electrostatic interactions of polar water molecules with electric fields generated by covalently bound pairs of opposite partial charge on the protein backbone. The methods allow calculation of the size of four hydration fractions: single water bridges, double water bridges, dielectric water clusters over polar-hydrophilic surfaces and water clusters over hydrophobic surfaces. These four fractions provide monolayer water coverage. The predicted SHM hydration fractions match closely measured hydration fraction values for collagen and for globular proteins. This report also presents water sorption findings that support the SHM. The SHM is applicable for cell systems where it has been studied. In seven cell systems studied, more than half of all of the cell water had properties unlike those of bulk water. The SHM predicts and explains the commonly cited and measured bound water fraction of 0.2-0.4 g of water/g of DM on proteins. The commonly accepted concept that water beyond this bound water fraction can be considered bulk-like water in its physical properties is unwarranted.     

The isolation of glycoproteins from bovine achilles tendon and their interaction with collagen

Two glycoprotein fractions, A and B, were isolated from bovine achilles tendon. Glycoprotein A was prepared from a 0.2m-sodium chloride extract and glycoprotein B was isolated from a 3m-magnesium chloride extract. They were free from serum proteins. Glycoprotein A was essentially free of collagen, but glycoprotein B contained about 8% collagen. Both glycoproteins gave several bands on isoelectric focusing. This technique was also used to demonstrate that both glycoprotein fractions interacted strongly with acid-soluble calf skin tropocollagen. It is concluded that these fractions are true components of tendon, and that they may have some function in the macromolecular organization of the tissue.     

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.     

Quantitative determination of the lipid peroxidation product 4-hydroxynonenal by high-performance liquid chromatography

4-Hydroxynonenal is a product formed in tissue and tissue fractions from polyunsaturated membrane lipids through a free radical-induced lipid peroxidation process. The biological properties of this aldehyde have been studied in many respects. This article describes for the first time a sensitive and reproducible method for quantitative analysis of 4-hydroxynonenal in biological samples as well as in lipid-containing foodstuffs. The method involves extraction of the aldehyde by dichloromethane from cells or microsomes trapped on an Extrelut column. Oils and foodstuffs are extracted with excess water. After additional sample cleanup by solid-phase extraction on a disposable octadecyl silica gel (ODS) extraction column, the sample is analyzed by high-performance liquid chromatography using an ODS column and methanol/water 65/35 (v/v) or acetonitrile/water 40/60 (v/v) as eluant; the detection wavelength is 220 nm. The method developed has a high precision with coefficients of variation of 1.4% (microsomes) to 3.5% (olive oil). The recovery depends on the sample type and lies between 45% (control microsomes) and 96% (solution of hydroxynonenal in water). The method has been used for the determination of 4-hydroxynonenal in microsomes, platelets, and various foodstuffs.     

SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.     

Sequential extracts of human bone show differing collagen synthetic rates

Type I collagen is the major bone protein. Little is known quantitatively about human bone collagen synthesis in vivo, despite its importance for the understanding of bone formation and turnover. Our aim was to develop a method that could be used for the physiological and pathophysiological investigation of human bone collagen synthesis. We have carried out preliminary studies in patients undergoing hip replacement and in pigs to validate the use of the flooding dose method using (13)C- or (15)N-labelled proline and we have now refined our techniques to allow them to be used in a normal clinical or physiological setting. The results show that the application of a flooding dose causes bone free-proline labelling to equilibrate with that of blood in pigs and human beings, so that only 150 mg of bone will provide enough sample to prepare and measure the labelling of three fractions of bone collagen (dissolved in NaCl, acetic acid and pepsin/acetic acid) which have the same relative labelling (1.0:0.43:0.1) as measured by GC-combustion-isotope ratio MS. The rates of incorporation were substantially faster than in skeletal muscle samples taken at the same time. The results suggest that different fractions of human bone collagen turnover at markedly higher rates than had been previously considered. This approach should allow us to discover how growth and development, food, activity and drugs affect bone collagen turnover and to measure the effects on it of ageing and bone disease.     

Quantification of collagen synthesis by cultured human glomerular cells

This study examines the amount of total collagen and its different fractions synthesized by cultured human glomerular epithelial and mesangial cells. Two quantitative techniques were used, namely estimation of proline (Pro) plus hydroxyproline (Hyp) present in the collagenase-sensitive proteins and ELISA or RIA of the different types of collagen. In addition, the pattern of collagen synthesis for both cell types was further examined using immunofluorescence methods and polyacrylamide gel electrophoresis. Glomerular epithelial cells synthesized mainly type IV collagen and it was, for the better part, cell-associated. Mesangial cells synthesized approx. 4-times more collagen than epithelial cells. Type I collagen was predominant, but there were also type IV and III collagens. Secreted and cell-associated collagens were present in roughly equivalent amounts. In both cell lines 10-14% of the newly synthesized collagen had been degraded within the cells. These results provide quantitative data on collagen synthesis by human glomerular cells in vitro and represent the first necessary stage before studying which factors mediate the development of glomerular sclerosis.