Fractionation and isolation of heparan sulfates using poly-L-lysine-Sepharose

A simple procedure for the isolation of heparan sulfates from pig lung using a poly-L-lysine-Sepharose column is described. Glycosaminoglycans are absorbed on poly-L-lysine-Sepharose at pH 7.5 and eluted with an NaCl linear gradient in the following order: hyaluronic acid (0.32 M NaCl), chondroitin (0.36 M NaCl), keratan sulfate (0.80 M NaCl), chondroitin 4-sulfate (0.86 M NaCl), chondroitin 6-sulfate (0.95 M NaCl), dermatan sulfate (0.91 M NaCl), heparan sulfate (1.2 M NaCl), and heparin (1.35 M NaCl). Based on these observations, isolation of heparan sulfate from pig lung crude heparan sulfate fractions which contain chondroitin sulfates and dermatan sulfate was attempted, using this chromatographic technique.     

Conformational behavior of chondroitin and chondroitin sulfate in relation to their physical properties as inferred by molecular modeling

Chondroitin and chondroitin sulfates belong to the family of glycosaminoglycans. They are most widely distributed in animal tissues, where they are involved in structural functions and in cell-cell communication. Their basic structures consist of a disaccharidic repeating unit of beta-D-glucuronic acid (GlcA) and 2-acetamido-2-deoxy-beta-D-galactose (GalNAc), this latter being sulfated at different positions. Molecular mechanics has been applied to calculate the adiabatic energy maps for each of the constituting disaccharides of chondroitin, chondroitin 4-sulfate, and chondroitin 6-sulfate using the MM3 force field. Based on these maps, higher levels of structural organization have been simulated. On one hand, the disordered state is studied through a Metropolis-based algorithm; the resulting chains present a behavior of semirigid polymers, with an order of stiffness: chondroitin 4-sulfate > chondroitin > chondroitin 6-sulfate. On the other hand, the exploration of the stable ordered forms leads to numerous helical conformations of comparable energies. Several of these conformations correspond to the experimentally observed ones. The ability of coordination with cations has also been explored, resulting in a preferential stereospecificity for calcium ions when compared to sodium ions.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epitope-specific changes in chondroitin sulfate/dermatan sulfate proteoglycans as markers in the lymphopoietic and granulopoietic compartments of developing bursae of Fabricius

The types and distributions of chondroitin sulfate proteoglycans within developing chick bursae of Fabricius were determined by indirect immunocytochemical analyses using mAb specific for chondroitin/dermatan sulfate epitopes. Analyses obtained from the use of well characterized mAb known to specifically identify chondroitin 4- and dermatan sulfates (antibody 2B6) and chondroitin 6-sulfate (antibody 3B3) were compared with those obtained from two additional mAb raised against chick chondroitin sulfates proteoglycans derived from hemopoietic tissue. The results indicate that chondroitin sulfate compositions of the adjacent lymphopoietic and granulopoietic compartments differ. Chondroitin 6-sulfate, notably absent from lymphopoietic regions, is a major chondroitin sulfate species in granulopoietic regions of day 13 bursae. Moreover, chondroitin 6-sulfate disappears from the granulopoietic compartment in a time course that corresponds to the decline in granulopoietic activity. Simultaneously, there is an apparent increase in chondroitin sulfates associated with developing medullary regions of lymphoid follicles. The content of chondroitin 4-/dermatan sulfates and, most significantly, of chondroitin/dermatan sulfates identified by antibodies raised against chick proteoglycans, increases within developing follicles. As a consequence, by day 18 of incubation, immunostained follicles become clearly demarcated from the connective tissue of the tunica propria. This study provides evidence that chondroitin sulfates are constituents of both lymphopoietic and granulopoietic microenvironments and that subtle changes occur within these proteoglycan structures during bursal development. These developmental changes in chondroitin sulfate compositions are consistent with these molecules playing a functional role in hemopoiesis.     

Structural characterization of the bovine tracheal chondroitin sulfate chains and binding of Plasmodium falciparum-infected erythrocytes

Sequestration of Plasmodium falciparum-infected red blood cells (IRBCs) in the human placenta is mediated by chondroitin 4-sulfate (C4S). A cytoadherence assay using chondroitin sulfate proteoglycans (CSPGs) is widely used for studying C4S-IRBC interactions. Bovine tracheal chondroitin sulfate A (CSA) preparation lacking a major portion of core protein has been frequently used for the assay. Here the CSPG purified from bovine trachea and CSA were assessed for IRBC binding and the CS chains studied in detail for structure-activity relationship. The IRBCs bound at significantly higher density to the CSPG than CSA. The CS chains of CSPG/CSA are heterogeneous with varying levels of 4- and 6-sulfates, which are distributed such that approximately 80% of the 4-sulfated disaccharides are present as single and blocks of two or three separated by one to three 6-sulfated disaccharides. The remainder of the 4-sulfated disaccharides is present in blocks composed of 4-12 units, separated by 6-sulfated disaccharides. In the IRBC adherence inhibition analysis, CSA fragments with 88%-92% 4-sulfate were significantly less inhibitory than the intact CSA, indicating that the regions consisting of shorter 4-sulfated blocks efficiently bind IRBCs despite the presence of relatively high levels of 6-sulfate. This is because the 6-sulfated disaccharides have unsubstituted C-4 hydroxyls that are crucial for IRBC binding. The presence of high levels of 6-sulfate, however, significantly interfere with the IRBC binding activity of CSA, which otherwise would more efficiently bind IRBCs. Thus our study revealed the distribution pattern of 4- and 6-sulfate in bovine tracheal CSA and structural basis for IRBC binding.     

Functional analysis of the chondroitin 6-sulfotransferase gene in relation to lymphocyte subpopulations, brain development, and oversulfated chondroitin sulfates.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. To obtain direct evidence regarding the function of C6ST and its product, chondroitin 6-sulfate, in vivo, we isolated the mouse C6ST gene (C6st) and generated mice deficient in this gene (C6st(-/-)) by embryonic stem cell technology. C6st(-/-) mice were born at approximately the expected frequency and were viable through adulthood. In the spleen of C6st(-/-) mice, the level of chondroitin 6-sulfate became almost undetectable. Analyses of these knockout mice provided insights into the biosynthesis of oversulfated chondroitin sulfates in mice; chondroitin sulfate D in the brain of null mice and the cartilage and telencephalon of null embryos disappeared, whereas the chondroitin sulfate E level in the spleen and brain of the null mice was unchanged. Despite the disappearance of chondroitin sulfate D structure, brain development was normal in the C6st(-/-) mice. Further analysis revealed that the number of CD62L(+)CD44(low) T lymphocytes corresponding to naive T lymphocytes in the spleen of 5-6-week-old C6st(-/-) mice was significantly decreased, whereas those in other secondary lymphoid organs were unchanged. This finding suggested that chondroitin 6-sulfate plays a role in the maintenance of naive T lymphocytes in the spleen of young mice.     

Effects of divalent cations on bovine testicular hyaluronidase catalyzed transglycosylation of chondroitin sulfates

Glycosaminoglycans were prepared as salts of different divalent cations and tested as donors in bovine testicular hyaluronidase catalyzed transglycosylation reactions. All of the metal cations examined had similar binding efficiency of divalent cations to hyaluronan. However, cations bound with different efficiencies to chondroitin sulfate species and the differences were marked in the case of chondroitin 6-sulfate; the numbers of cations bound per disaccharide unit were estimated to be 0.075 for Mn, 1.231 for Ba, 0.144 for Zn, and 0.395 for Cu. While barium salt of chondroitin sulfates enhanced transglycosylation, the zinc salt of chondroitin sulfates inhibited transglycosylation. Therefore, by selecting the proper divalent cation salt of chondroitin sulfates as a donor in the transglycosylation reaction it is possible to improve the yields of the products.     

Sulfated glycosaminoglycans of human aorta: chondroitin 6-sulfate increase with age.

The proportions of chondroitin 4 and 6 sulfates of intima + media layers of normal human aortae vary with age. The two isomers are in approximately equal amounts in aortae of young individuals, while the 6-sulfate is more abundant in those of adult individuals. This increase of chondroitin 6-sulfate is even more pronounced for intima + media obtained from atherosclerotic aortae.     

Developmental distribution of glycosaminoglycans in embryonic rat brain: Relationship to axonal tract formation

Glycosaminoglycans, the sugar moieties of proteoglycans, modulate axonal growth in vitro. However, their anatomical distribution in relation to developing axonal tracts in the rat brain has not been studied. Here, we examined the immunohistochemical distribution of chondroitin-6-sulfate and chondroitin-4-sulfate, two related glycosaminoglycan epitopes, which are present in three types of glycosaminoglycans: chondroitin sulfate C, chondroitin sulfate A, and chondroitin sulfate B. Further, we compared their distribution pattern to that of axonal tract development. Both glycosaminoglycan epitopes showed a heterogeneous spatiotemporal distribution within the developing rat brain. However, the expression of chondroitin-4-sulfate was more restricted than that of chondroitin-6-sulfate, although both epitopes were detected from embryonic day 13 until the day of birth, overlapping in many regions of the central nervous system including cortex, hippocampus, thalamus, and hindbrain. After birth, the levels of expression of both glycosaminoglycan epitopes progressively decreased and were practically undetectable after the first postnatal week. The expression of chondroitin-6-sulfate and, to a lesser extent, that of chondroitin-4-sulfate, was preferentially associated to the extracellular matrix surrounding specific axon bundles. However, the converse association was not true, and several apparently similar types of axon developed on a substrate devoid of both types of glycosaminoglycan epitopes. These results provide an anatomical background for the idea that different types of glycosaminoglycans may contribute to establish the complex set of guidance cues necessary for the specific development of defined axon tracts in the central nervous system. © 1996 John Wiley & Sons, Inc.     

Sulfated mucopolysaccharides of midgestation embryonic and extraembryonic tissues of the mouse

Incorporation of [³⁵S]sulfate into sulfated mucopolysaccharides has been characterized in midgestation mouse embryo, yolk sac, trophoblast, and decidua. Enzymatic analysis indicated that chondroitin sulfates contained approximately half of the label in embryo, trophoblast, and decidua, but less than 20% in yolk sac. While the labeled chondroitin sulfate fraction of trophoblast and decidua was mainly chondroitin-4-sulfate, only embryo contained a significant proportion of labeled chondroitin-6-sulfate. The relative incorporation into embryo chondroitin-6-sulfate was also substantially higher than that observed in four adult soft tissues. Labeled dermatan sulfate was absent from the embryo and yolk sac, but small amounts might have been synthesized by the placenta. Nitrous acid degradation studies revealed that essentially all the chondroitinase resistant MPS was N-sulfated, i.e., heparan sulfate and/or heparin. Electrophoretic profiles indicate that the bulk of the N-sulfated material resembles heparan sulfate rather than heparin. Electrophoretic heterogeneity and slow migration rates relative to standard markers suggest that the majority of labeled chondroitin sulfates may be undersulfated. The different mucopolysaccharide patterns in the various tissues may reflect their specialized properties and functions.     

Immunoelectron microscopic analysis of chondroitin sulfates during calcification in the rat growth plate cartilage

The proximal growth plate cartilage of rat tibia was fixed in the presence of ruthenium hexamine trichloride (RHT) in order to preserve proteoglycans in the tissue. Quantitative changes of chondroitin sulfates during endochondral calcification were investigated by immunoelectron microscopy using mouse monoclonal antibodies 1-B-5, 2-B-6, and 3-B-3, which recognize unsulfated, 4-sulfated, and 6-sulfated chondroitin sulfates, respectively. The content of chondroitin-4-sulfate in the cartilage matrix increased from the proliferative zone to the calcifying zone, while that of unsulfated chondroitin sulfate decreased. Chondroitin-6-sulfate remained constant from the proliferative zone to the upper hypertrophic zone, then decreased in the calcifying zone. The immunoreaction to each antibody increased conspicuously in the cartilagenous core of metaphysial bone trabeculae. The changes of sulfation in chondroitin sulfate chains of proteoglycans may play an important role in inducing and/or promoting calcification in growth plate cartilage.     

Divergent regulation of proteoglycan and glycosaminoglycan free chain expression in human keratinocytes and melanocytes

Summary Keratinocytes and melanocytes, which together form units of structure and function within human epidermis, are known to differ in expression of autocrine growth factors, particularly those with heparin binding affinity. Because such cytokines could be regulated by the endogenous heparinlike glycosaminoglycan, heparan sulfate, proteoglycan synthesis was compared between human keratinocytes and melanocytes cultured from a common donor. Following steady-state isotopic labeling under conditions of active growth (low density cultures) and growth inhibition (high density cultures), the sulfated polymers were isolated from conditioned media and cell extracts. We found that keratinocytes produced substantially more sulfated glycosaminoglycans than did the melanocytes. There was no evidence for hyaluronic acid synthesis by the melanocytes. The majority of [³⁵S]-sulfate labeling was in the heparan sulfates of the keratinocytes and in the chondroitin sulfates of the melanocytes. During the transition from active growth to growth inhibition, there was increased heparan sulfate proteoglycan and free chain synthesis by keratinocytes but not by melanocytes, and chondroitin sulfate proteoglycan production declined in both cell lineages. The differences may reflect divergent evolution as each cell type came to exploit those complex polysaccharides in different ways to regulate molecular pathways of growth and differentiation. The coupling of growth inhibition with augmented synthesis of heparan sulfates observed for the keratinocytes suggests a regulatory role in growth factor signaling in that cell type.     

A fingerprinting method for chondroitin/dermatan sulfate and hyaluronan oligosaccharides

A previously published method for the analysis of glycosaminoglycan disaccharides by high pH anion exchange chromatography (Midura,R.J., Salustri,A., Calabro,A., Yanagishita,M. and Hascall,V.C. (1994), Glycobiology,4, 333-342) has been modified and calibrated for chondroitin and dermatan sulfate oligosaccharides up to hexasaccharide in size and hyaluronan oligosaccharides up to hexadecasaccharide. For hyaluronan oligosaccharides chain length controls elution position; however, for chondroitin and dermatan sulfate oligosaccharides elution times primarily depend upon the level of sulfation, although chain length and hence charge density plays a role. The sulfation position of GalNAc residues within an oligosaccharide is also important in determining its elution position. Compared to 4-sulfation a reducing terminal 6-sulfate retards elution; however, when present on an internal GalNAc residue it is the 4-sulfate containing oligosaccharide which elutes later. These effects allow discrimination between oligosaccharides differing only in the position of GalNAc sulfation. Using this simple methodology, a Dionex CarboPac PA-1 column with NaOH/NaCl eluents and detection by absorbance at 232 nm, a quantitative analytical fingerprint of a chondroitin/dermatan sulfate chain may be obtained, allowing a determination of the abundance of chondroitin sulfate, dermatan sulfate, and hyaluronan along with an analysis of structural features with a linear response to approximately 0.1 nmol. The method may readily be calibrated using either commercial disaccharides or the di- and tetrasaccharide products of a limit digest of commercial chondroitin sulfate by chondroitin ABC endolyase. Commercially available and freshly prepared shark, whale, bovine, and human cartilage chondroitin sulfates have been examined by this methodology and we have confirmed that freshly isolated shark cartilage CS contains significant amounts of the biologically important GlcA2Sbeta(1-3)GalNAc6S structure.     

Identification of Specific Chondroitin Sulfate Species in Cutaneous Autoimmune Disease

Cutaneous lupus erythematosus and dermatomyositis (DM) are chronic inflammatory diseases of the skin with accumulated dermal mucin. Earlier work has shown chondroitin sulfate (CS) accumulation within the dermis of discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and DM lesions compared with control skin. Immunohistochemistry for C4S revealed a greater density in DLE and DM lesions, whereas SCLE lesions did not differ from controls. Scleredema and scleromyxedema are attributed to increased hyaluronic acid, and lesional samples from these diseases also demonstrated accumulated dermal C4S. Interferon-γ and interleukin-1α, but not interferon-α, treatment of cultured dermal fibroblasts induced mRNA expression of CHST-11, which attaches sulfates to the 4-position of unsulfated chondroitin. These studies on possible CS core proteins revealed that serglycin, known to have C6S side chains in endothelial cells, had greater density within DM dermal endothelia but not in DLE or SCLE, following the pattern of C6S overexpression reported previously. CD44 variants expand the CS binding repertoire of the glycoprotein; CD44v7 co-localized to the distribution of C4S in DLE lesions, a finding not observed in DM, SCLE lesions, or controls. Because C4S and C6S have immunologic effects, their dysregulation in cutaneous mucinoses may contribute to the pathogenesis of these disorders.     

Structure of chondroitin sulfates analyses of the products formed from chondroitin sulfates A and C by the action of the chondroitinases C and AC from Flavobacterium heparinum

The structures of chondroitin sulfate A from whale cartilage and chondroitin sulfate C from shark cartilage have been examined with the aid of the chondroitinases AC and C from Flavobacterium heparinum. The analyses of the products formed from the chondroitin sulfates by the action of the chondroitinases have shown that three types of oligosaccharides compose the structure of chondroitin sulfate A, namely, a dodeca-, hexa- and a tetra-saccharide, containing five, two and one 4-sulfated disaccharides per 6-sulfated disaccharide residue, respectively. The polymer contains an average of 3 mol of each oligosaccharide per mol of chondroitin sulfate A. Each mol of chondroitin sulfate C contains an average of 5 mol of 4-sulfated disaccharide units. A tetra-saccharide containing one 4-sulfated disaccharide and one 6-sulfated disaccharide was isolated from this mucopolysaccharide by the action of the chondroitinase C, indicating that the 4-sulfated disaccharides are not linked together in one specific region but spaced in the molecule.     

Mucopolysaccharide-polypeptide interactions: Effect of the position of the sulfate group

The differences in the interaction in solution of poly(l-lysine) with chondroitin 6-sulfate (chondroitin sulfate C) and with chondroitin 4-sulfate (chondroitin sulfate A) have been studied by circular dichroism spectroscopy. Both mucopolysaccharides force the poly(l-lysine) to adopt the α-helix in solution rather than the charged coil form expected at neutral pH. The observed spectra indicates that the polypeptide is at least 80% helical when the 6-sulfate form is present, but only about 20% α-helical in the presence of chondroitin 4-sulfate. Thus chondroitin66-sulfate has a stronger conformation directing effect on poly(l-lysine) than does the 4-sulfate, which is probably due to the different positions of the sulfate group on the polysaccharide c chain.     

Plasmodium falciparum: Assessment of parasite-infected red blood cell binding to placental chondroitin proteoglycan and bovine tracheal chondroitin sulfate A

In pregnant women infected with Plasmodium falciparum, the infected red blood cells (IRBCs) sequester in placenta by binding to the chondroitin 4-sulfate (C4S) chains of low sulfated chondroitin sulfate proteoglycan (CSPG). Placental CSPG, the natural receptor for IRBC adherence in the placenta, is the ideal molecule for studying structural interactions in IRBC adhesion to C4S, adhesion inhibitory antibody responses, and identification of parasite adhesive protein(s). However, because of difficulty involved in purifying placental CSPG, the commercially available bovine tracheal chondroitin sulfate A (bCSA), a copolymer having structural features of both C4S and C6S, has been widely used. To determine the validity of bCSA for C4S-IRBC interaction studies, we comparatively evaluated the characteristics of IRBC binding to placental CSPG and bCSA using three commonly used parasite strains. The results indicate that, in all three parasites studied, the characteristics of IRBC binding to placental CSPG and bCSA are qualitatively similar, but the binding capacity with respect to both the number of IRBCs bound per unit area of coated surface and binding strength is significantly higher for CSPG than bCSA regardless of whether parasites were selected on CSPG or bCSA. These results demonstrate that placental CSPG is best suited for studying interactions between parasite adhesive protein(s) and C4S, and have implications in understanding C4S-IRBC structural interactions.     

The application of high-performance liquid chromatography in enzymatic assays of chondroitin sulfate isomers in normal human urine

A study of the urinary excretion of isomeric chondroitin sulfates in normal individuals by high-performance liquid chromatographic (HPLC) determinations of the unsaturated disaccharides produced by digestion with chondroitinases is described. The composition of the HPLC mobile phase was systematically varied in order to select the optimal conditions for separation.The data show that chondroitin 4-sulfate is the major component of the chondroitin sulfate isomers in normal urine, and that chondroitin 6-sulfate is a lesser component. It is also evident that dermatan sulfate is present in small quantities in normal urine.     

Selective hydrolysis of chondroitin sulfates by hyaluronidase

Chondroitin 4-sulfate and chondroitin 6-sulfate were incubated with testicular hyaluronidase in the presence of excess beta-glucuronidase. The beta-glucuronidase caused rapid removal of the nonreducing terminal beta-D-glucuronosyl residues from the oligosaccharides formed by the action of the hyaluronidase, destroying the oligosaccharide acceptors required for the transglycosylation activity of hyaluronidase and releasing free D-glucuronic acid at a rate that was equal to the rate of the hyaluronidase-catalyzed hydrolysis. When hyaluronidase was assayed at 37 degrees C in the presence of 0.05 M NaCl, 0.05 M Na2SO4, and 0.1 M sodium acetate at pH 5, chondroitin 4-sulfate was hydrolyzed at 1.5 times the rate found for chondroitin 6-sulfate. When hyaluronidase was assayed at 45 degrees C in 0.06 M sodium acetate at pH 6, chondroitin 4-sulfate was hydrolyzed at 8 times the rate observed for chondroitin 6-sulfate. Under the pH5 conditions, the chondroitin 4-sulfate was converted to a mixture of tri- and pentasaccharides, while the chondroitin 6-sulfate was converted primarily to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide. Under the pH 6 conditions, the chondroitin 4-sulfate was converted to a mixture of penta- and heptasaccharides, with only a small amount of trisaccharide, but the products from chondroitin 6-sulfate were a mixture of oligosaccharides ranging in degree of polymerization from 7 to 25 monosaccharides per oligosaccharide. End-group analyses of the products formed at pH 6 showed that both substrates were cleaved preferentially at the glycosidic bonds of the 4-sulfated disaccharides.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fe and Fe initiated peroxidation of sonicated and non-sonicated liposomes made of retinal lipids in different aqueous media

Retina is highly susceptible to oxidative damage due to its high content of polyunsaturated fatty acids (PUFAs), mainly docosahexaenoic acid (22:6 n3). Lipid peroxidation process is thought to be involved in many physiological and pathological events. Many model membranes can be used to learn more about issues that cannot be studied in biological membranes. Sonicated liposomes (SL) and non-sonicated liposomes (NSL) prepared with lipids isolated from bovine retina and characterized by dynamic light-scattering, were submitted to lipid peroxidation, under air atmosphere at 22 °C, with Fe²⁺ or Fe³⁺ as initiator, in different aqueous media. Conjugated dienes and trienes, determined by absorption at 234 and 270 nm respectively, and thiobarbituric acid-reactive substances were measured as a function of time. Peroxidation of SL or NSL initiated with 25 μM FeSO₄ in 20 mM Tris-HCl pH 7.4 resulted in an increase in TBARS production after a lag phase of 60 min. Incubation of both types of liposomes in water resulted in shortening of the lag phase at 30 min. When lipid peroxidation was performed in 0.15 M NaCl, lag phase completely disappeared. On the other hand, FeCl₃ (25 μM) induced a limited production of TBARS only just after 30 min of incubation. When Fe²⁺- or Fe³⁺-lipid peroxidation of both types of liposomes was carried out in water or 0.15 M NaCl, formation of conjugated dienes and conjugated trienes were higher than in reactions carried out in 20 mM Tris-HCl pH 7.4.Our results established that both liposome types were susceptible to Fe²⁺- and Fe³⁺-initiated lipid peroxidation. However, Fe²⁺ showed a clearly enhanced effect on peroxidation rate and steady state concentration of oxidation products.We verified that peroxidation of liposomes made of retinal lipids is affected not only by type of initiator but also by aqueous media. This model constitutes a useful system to study formation of lipid peroxidation intermediaries and products in an aqueous environment.