Chemistry of the collagen cross-links. Origin and partial characterization of a putative mature cross-link of collagen.

The conversion of the reducible divalent cross-links in collagen to non-reducible multivalent cross-links in mature collagen has resulted in the identification of several new amino acids as the putative mature cross-link. None of these compounds has completely satisfied the necessary criteria. We have now isolated an amino acid of high Mr, derived from lysine, that is only present in high-Mr peptides derived from mature collagen. Its increase with age of the tissue correlates with the decrease in the reducible cross-links, and it is present both in mature skin and bone, which are initially cross-linked through the aldimine and oxo-imine divalent cross-link respectively. We propose that this amino acid, as yet incompletely characterized and designated compound M, is a major cross-link of mature collagen.     

Oxidative deamination of epsilon-aminolysine residues and formation of Schiff base cross-linkages in cell envelopes of Escherichia coli.

Oxidative deamination of the epsilon-amino group of lysyl residues to form allysine is the initial reaction in the cross-linking of collagen and elastin in vertebrates. The allysyl residues, generated by lysyl oxidase in this reaction, condense with either other allysyl residues or epsilon-amino groups of lysyl or hydroxylysyl to form aldol or Schiff base cross-links. This paper presents evidence that similar allysyl residues and Schiff base cross-links are synthesized in cell envelopes of Escherichia coli. Acid hydrolysis followed by amino acid analysis of envelopes either reduced with NaB[3H]4 or labeled with [14C]lysine and reduced with NaBH4 yielded allysine and two labeled fragments with elution profiles and molecular weights (250 and 330) consistent with Schiff base products derived at least in part from allysine. When [6-3H]lysine-labeled cell envelopes were incubated at 37 degrees C, gradual release of tritiated water occurred. This suggests that an enzymatic reaction catalyzes the deamination of lysine in E. coli membranes and that the higher molecular weight proteins detected in stationary phase or in log phase cell envelopes after NaBH4 reduction occur as a result of formation of Schiff base cross-links.     

Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures

Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.     

Collagen cross-linking. Effect of D-penicillamine on cross-linking in vitro.

D-Pencillamine is believed to inhibit collagen cross-link biosynthesis by forming thiazolidine rings with lysyl-derived aldehydes that are intermediates in bifunctional cross-link synthesis. Recently, we showed that aldehyde biosynthesis catalyzed by lysyl oxidase occurs after the onset of fibril formation and that nascent aldehydes form Schiff-base cross-links rapidly in fibrils. This suggested that the accessibility of D-penicillamine to most aldehydes formed during cross-link synthesis might be limited. To study this, reconstituted chick bone collagen fibrils were incubated in vitro with highly purified lysyl oxidase and D-penicillamine. As reported in previous studies in vivo, allysine content increased and polyfunctional cross-link synthesis decreased with D-penicillamine. However, the concentration of bifunctional cross-links increased rather than decreased due to a 2-fold increase in N6:6'-dehydro-5,5'-dihydroxylysinonorleucine. Hydroxyallysine, an intermediate in formation of this Schiff base, decreased. A time study indicated that allysine levels increased primarily after the bulk of Schiff base synthesis. These results indicate that D-penicillamine does not inhibit bifunctional cross-link synthesis as previously suggested. Its principal effect is to block synthesis of polyfunctional cross-link products from Schiff base cross-link precursors and to cause accumulation of these precursors. This effect may be due to interference with the close molecular packing required for polyfunctional cross-link synthesis. These results also suggest a mechanism for the relative insensitivity of tissues such as bone with high hydroxylysine content to D-penicillamine. In this study, D-penicillamine caused selective accumulation of allysyl and not hydroxyallysyl residues. In bone as opposed to soft tissues, hydroxyallysyl residues are intermediates in synthesis of almost all cross-links.     

Cross-linking methionine and amine residues with reactive halogen species

Irreversible cross-links are increasingly being recognized as important posttranslational oxidative protein modifications that contribute to tissue injury during oxidative stress and inflammation. They also have a structural function in extracellular matrix proteins such as collagen IV. Likely contenders for forming such cross-links are the reactive halogen species that are generated by neutrophils and eosinophils, including hypochlorous acid, hypobromous acid, and their related haloamines. Methionine residues are kinetically preferred targets for these oxidants and oxidation can potentially result in sulfilimine (>SN–) bonds with amines. Therefore, we investigated whether oxidation of methionine in the model peptide formyl-Met-Leu-Phe-Lys (fMLFK) produces cross-links with lysine residues, using mass spectrometry to characterize the products. As expected, the sulfoxide was the major product with each reactive halogen species. However, intra- and intermolecular cross-linked products were also formed. Isomers of an intramolecular sulfilimine were readily produced by hypobromous acid and bromamines, with hypochlorous acid forming lesser amounts. The predominant cross-link with chloramines was an intermolecular bond between the sulfur of fMLFK and the amine derived from the chloramine. Reactive halogen species also formed these sulfilimine cross-links in other peptides that contain methionine. We propose that protein cross-links involving methionine and amine residues will form via this mechanism when granulocytes are activated at sites of inflammation. Our results also support the proposal that reactive halogen species generated by the peroxidase peroxidasin could be responsible for the sulfilimine bonds that are integral to the structure of collagen IV.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

The chemistry of the collagen cross-links. Age-related changes in the reducible components of intact bovine collagen fibres

The change in the amounts of the three major reducible cross-links was followed throughout the bovine-life span. The major reducible cross-link in embryonic skin is 6,7-dehydro-N(epsilon) -(2-hydroxy-5-amino-5-carboxypentyl)hydroxylysine, but this is gradually replaced in the latter stages of gestation or early postnatal growth period by two other Schiff bases, 6,7-dehydro-N(epsilon)-(5-amino-5-carboxypentyl)hydroxylysine and a component not yet identified, designated Fraction C. These latter two Schiff bases increase in amount during the rapid growth period to a maximum, after which they then slowly decrease until at maturity they are virtually absent. The proportion of these Schiff bases closely reflects the rate of growth, i.e. the amount of newly synthesized collagen present at any one time. Similarly, the three Schiff bases present in tendon and the one in cartilage slowly decrease during maturation. No evidence for the possible stabilization of these aldimine bonds during maturation by reduction in vivo was found by three different analytical techniques. Concurrently with the decrease in the proportion of the Schiff bases some new reducible components increased during maturation, but their characterization as N(epsilon)-glycosylamines demonstrated that they were not related to the lysine-derived aldehyde components. The significance of these components in the aging process cannot at present be assessed. As no evidence was obtained for any new reducible cross-links replacing the Schiff bases, it is probable that the latter are intermediate cross-links and that during maturation they are stabilized to some as yet unknown non-reducible cross-link as previously proposed (Bailey, 1968).     

Intermolecular cross-linking and stereospecific molecular packing in type I collagen fibrils of the periodontal ligament

A trypsin digest of denatured NaB3H4-reduced native bovine periodontal ligament was prepared and fractionated by gel filtration and cellulose ion-exchange column chromatography. Prior to trypsin digestion, a complete acid hydrolysate was subjected to analyses for nonreducible stable and reducible intermolecular cross-links. Minute amounts of the former and significant amounts of the reduced cross-links dihydroxylysinonorleucine (1.1 mol/mol of collagen), hydroxylysinonorleucine (0.9 mol/mol of collagen), and histidinohydroxymerodesmosine (0.6 mol/mol of collagen) were found. The covalent intermolecular cross-linked two-chained peptides that were isolated were subjected to amino acid and sequence analyses. The structures for the different two-chained linked peptides were alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6-(993-22c)[Lysald-16c], alpha 1CB4-5(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c], alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Lysald-16c], and alpha 2CB4(76-90)[Hyl-87] X alpha 1CB6(993-22c)[Hylald-16c]. The cross-link in each peptide was glycosylated. This is the first characterization by sequence analysis of a cross-link involving Hyl-87 in an alpha 2 chain in collagen. A stoichiometric conversion of residue 16c aldehyde to an intermolecular cross-link in each of the COOH-terminal nonhelical peptide regions of both alpha 1 chains in a molecule of type I collagen was found. The ratio of alpha 1 to alpha 2 intermolecularly cross-linked chains involved was 3.3:1, indicating a stereospecific three-dimensional molecular packing of type I collagen molecules in bovine periodontal ligament.     

Isolation and structure of a cross-linked tripeptide from calf bone collagen.

A cross-linked tripeptide has been isolated from alkaline hydrolysates of NaB3H4-reduced calf bone collagen. The peptide contains dihydroxylysinonorleucine, the most abundant cross-link in bone collagen, and it has a single N-terminal proline and a single C-terminal valine. These amino acids are in peptide linkage with the cross-link, in a trans configuration with respect to the secondary amine.     

The influence of 1-hydroxyethane-1,1-diphosphonate and dichloromethanediphosphonate on lysine hydroxylation and cross-link formation in rat bone, cartilage and skin collagen.

The effects in vivo of dichloromethanediphosphonate and 1-hydroxyethane 1,1-diphosphonate on collagen solubility, hydroxylation of lysine and proline and on the formation of collagen intermolecular cross-links were studied by using rat bone, cartilage and skin tissues. Dichloromethanediphosphonate decreased bone collagen solubility both in acetic acid and after pepsin treatment. Although none of the diphosphonates had any effect on the hydroxylation of proline, dichloromethane-diphosphonate, but not 1-hydroxyethane-1,1-diphosphonate, increased the number of hydroxylysine residues in the alpha-chains of bone, skin and cartilage collagen. The stimulatory effect was dose-dependent. The dichloromethanediphosphonate-mediated increase in hydroxylysine residues in bone and cartilage was manifested in an increase of dihydroxylysinonorleucine, the cross-link that is formed by the condensation of two hydroxylysine residues. The cross-link hydroxylysinonorleucine, a condensation product of hydroxylysine and lysine, on the other hand, was decreased. The total number of intermolecular cross-links was not changed by the diphosphonate.     

Collagen cross-linking. Synthesis of collagen cross-links in vitro with highly purified lysyl oxidase.

In this paper, the synthesis of collagen cross-links in vitro was investigated in a defined system consisting of highly purified chick cartilage lysyl oxidase and chick bone collagen fibrils. Cross-link synthesis in vitro was quite similar to the biosynthesis of collagen cross-links in vivo. Enzyme-dependent synthesis of cross-link intermediates and cross-linked collagen derived from lathyritic collagen occurred. The concentration of the two principal reducible cross-links, N6:6'-dehydro-5,5'-dihydroxylysinonorleucine and N6:6'-dehydro-5-hydroxylysinonorleucine, increased to a peak value of approximately two cross-links per molecule and then decreased. Synthesis of histidinohydroxymerodesmosine and a second polyfunctional cross-link of unknown structure began after synthesis of bifunctional cross-links was largely completed and proceeded linearly afterwards. Inhibition of lysyl oxidase after the bulk of bifunctional cross-link synthesis had occurred did not alter the rate of decrease in reducible cross-link concentration but did inhibit further histidinohydroxymerodesmosine synthesis. These results indicate that lysyl oxidase and collagen fibrils are the only macromolecules required for cross-link biosynthesis in vivo. It is likely that the decrease in reducible cross-links observed during fibril maturation results from spontaneous reactions within the collagen fibril rather than additional enzymatic reactions.     

The chemistry of the collagen cross-links. A convenient synthesis of the reduction products of several collagen cross-links and cross-link precursors

New synthetic routes to the reduction products of several collagen cross-links and cross-link precursors are described. By the use of these routes hydroxynorleucine, 5,6-dihydroxynorleucine, hydroxylysinonorleucine and hydroxylysinohydroxynorleucine can be synthesized via one common synthetic intermediate. The synthetic routes provide a convenient source of these unusual amino acids, as well as confirming the structure of hydroxylysinohydroxynorleucine and the other lysine-derived residues found in borohydride-reduced collagens.     

Lysinonorleucine cross-link formation in alpha amino heptenoic acid-substituted peptide derivatives

Studies on the cross-linking of a tripeptide (t-butyloxycarbonyl-L -alanyl-D ,L -2-amino-6-heptenoyl-L -alanine methyl ester) have shown that it is possible to form specific cross-links in good yields through Schiff base formation of the ε amino group of lysine. The heptenoic acid residue has been ozonized to an aldehyde and condensed with the ε amino of lysine in the compounds alpha-t-butyloxycarbonyl-L -alanyl-L -lysine methyl ester and alpha-t-butyloxycarbonyl-L -lysine methyl ester to form the cross-link, lysinonorleucine. This compound has been stabilized by reduction with sodium borohydride and quantitated on the amino acid analyzer. This technique converts from 60 to 98% of the available aldehyde to lysinonorleucine.     

Stable, nonreducible cross-links of mature collagen.

During in vivo maturation, and also during in vitro incubation with physiological buffers, native collagen fibers display a progressive increase in tensile strength and insolubility. Paralleling these physiologically important changes is a progressive loss of the reducible cross-links which initially join the triple-chained subunits of collagen fibers. Although there is evidence suggesting that the reducible cross-links are gradually transformed into more stable, nonreducible cross-links during maturation, the nature of the transformation process and the structure of the stable "mature" cross-links has remained a mystery. In order to test the possibility that cross-link transformation involves addition of a nucleophilic amino acid residue to the reducible cross-links, histidine, arginine, glutamate, aspartate, lysine, and hydroxylysine residues were chemically modified, and the effect of each modification procedure on the in vitro transformation of reducible cross-links was ascertained. The results of these experiments indicated that destruction of histidine, arginine, glutamate, and aspartate residues has no measurable effect on the rate and extent of reducible cross-link transformation in hard tissue collagens. In contrast, modification of lysine and hydrocylysine residues with a wide variety of specific reagents completely blocks the transformation of reducible cross-links. Removal of the reversible blocking groups from lysine and hydroxlylysine residues then allows the transformation to proceed normally. These results indicate that collagen maturation involves nucleophilic addition of lysine and/or hydroxylysine residues to the electrophilic double bond of the reducible cross-links, yielding derivatives which are not only more stable but also capable of cross-linking more collagen molecules than their reducible precursors.     

Reactions of lipid-derived malondialdehyde with collagen.

Malondialdehyde is a product of fatty acid oxidation (e.g. from low density lipoprotein) implicated in the damage of proteins such as collagen in the cardiovascular system (Chio, K. J., and Tappel, A. L. (1969) Biochemistry 8, 2821-2827). Its concentration is raised in diabetic subjects probably as a side effect of increased protein glycation. Collagen has enzyme-catalyzed cross-links formed between its individual molecules that are essential for maintaining the structure and flexibility of the collagen fiber. The cross-link dehydro-hydroxylysinonorleucine reacts irreversibly with 10 mM malondialdehyde at least 3 orders of magnitude faster than glucose reactions with lysine or arginine, such that there is little cross-link left after 1 h at 37 degrees C. Other cross-links and glycated elements of collagen are also vulnerable. Several possible products of malondialdehyde with collagen cross-links are proposed, and the potential involvement of collagenous histidine in these reactions is discussed. We have also isolated Ndelta-(2-pyrimidyl)-L-ornithine from collagenous arginine reacted with malondialdehyde. The yields of this product were considerably higher than those from model reactions, being approximately 2 molecules/collagen molecule after 1 day at 37 degrees C in 10 mM malondialdehyde. Collagenous lysine-derived malondialdehyde products may have been present but were not protected from protein acid hydrolysis by standard reduction techniques, thus resulting in a multitude of fragmented products.     

Subunit structure and assembly of the globular domain of basement-membrane collagen type IV

The globular domain of collagen IV was solubilized by collagenase digestion from a mouse tumor, human placenta and bovine aorta and was purified by chromatographic methods. The materials show a unique, mainly non-collagenous amino acid composition and contain small amounts of glucosamine and galactosamine. The globular structures with Mr = 170 000 appear as a hexameric assembly originating from two collagen IV molecules. Subunits of this assembly are two different dimers Da and Db (Mr about 56 000) and monomers (Mr = 28 000). Their N-terminal amino acid sequences start with short triple-helical sequences, which overlap with the C-terminal triple helix of the alpha 1(IV) and alpha 2(IV) chain, demonstrating that the globule originates from the C terminus of collagen IV. Dimers arise from monomers by disulfide cross-linking (form Db) and/or formation of non-reducible cross-links (form Da). Reduction under non-denaturing conditions causes partial dissociation of the globule and of collagen IV dimers, indicating that reducible cross-links are formed between monomers of two different collagen IV molecules. Dissociation of the hexamer into the subunits can be achieved with 8 M urea, sodium dodecyl sulfate or in the pH range 2.5-4. The latter indicates that carboxyl groups are essential for association. Mixtures of the subunits (monomers and dimers) or purified dimers reassemble in neutral buffer into hexamers as shown by ultracentrifugation and electron microscopy. Reconstituted hexamers, however, dissociate in a much broader pH range than the native globules. Circular dichroic spectra indicate that the structure is more completely refolded from acid-treated than from urea-treated material. These data suggest that globules originating from monomers (as existing in single collagen IV molecules) are stabilized by the adjacent triple helix. Covalent cross-link formation stabilizes the globular structure and allows reconstitution in stoichiometric proportions.     

Collagen cross-linking. Purification and substrate specificity of lysyl oxidase.

Lysyl oxidase is a specific amine oxidase that catalyzes the formation of aldehyde cross-link intermediates in collagen and elastin. In this study, lysyl oxidase from embryonic chick cartilage was purified to constant specific activity and a single protein band on sodium dodecyl sulfate acrylamide gel electrophoresis. This band had an apparent molecular weight of 62,000. The eluted protein cross-reacted with inhibiting antisera developed against highly purified lysyl oxidase. The highly purified enzyme was active with both insoluble elastin and embryonic chick skin or bone collagen precipitated as reconstituted, native fibrils. There was low activity with nonhydroxylated collagen, collagen monomers, or native fibrils isolated from lathyritic calvaria. The maximum number of aldehyde intermediates formed per molecule of collagen that became insoluble was two. These results indicate that lysyl oxidase has maximum activity on ordered aggregates of collagen molecules that may be overlapping associations of only a few collagen molecules across. Formation of aldehyde intermediates and cross-links during fibril formation may facilitate the biosynthesis of stable collagen fibrils and contribute to increased fibril tensile strength in vivo.     

Determination of the relationship between collagen cross-links and the bone-tissue stiffness in the porcine mandibular condyle

Although bone-tissue stiffness is closely related to the degree to which bone has been mineralized, other determinants are yet to be identified. We, therefore, examined the extent to which the mineralization degree, collagen, and its cross-links are related to bone-tissue stiffness. A total of 50 cancellous and cortical bone samples were derived from the right mandibular condyles of five young and five adult female pigs. The degree of mineralization of bone (DMB) was assessed using micro-computed tomography. Using high-performance liquid chromatography, we quantified the collagen content and the number of cross-links per collagen molecule of two enzymatic cross-links: hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP), and one non-enzymatic cross-link: pentosidine (Pen). Nanoindentation was used to assess bone-tissue stiffness in three directions, and multiple linear regressions were used to calculate the correlation between collagen properties and bone-tissue stiffness, with the DMB as first predictor. Whereas the bone-tissue stiffness of cancellous bone did not differ between the three directions of nanoindentation, or between the two age groups, cortical bone-tissue stiffness was higher in the adult tissue. After correction for DMB, the cross-links studied did not increase the explained variance. In the young group, however, LP significantly improved the explained variance in bone-tissue stiffness. Approximately half of the variation in bone-tissue stiffness in cancellous and cortical bone was explained by the DMB and the LP cross-links and thus they cannot be considered the sole determinants of the bone-tissue stiffness.     

Chlorination of pyridinium compounds. Possible role of hypochlorite, N-chloramines, and chlorine in the oxidation of pyridinoline cross-links of articular cartilage collagen type II during acute inflammation

Reactive oxygen species produced by activated neutrophils and monocytes are thought to be involved in mediating the loss of collagen and other matrix proteins at sites of inflammation. To evaluate their potential to oxidize the pyridinoline (Pyd) cross-links found in collagen types I and II, we reacted hydrogen peroxide (H(2)O(2)), hypochlorous acid/hypochlorite (HOCl/OCl(-)), and singlet oxygen (O(2)((1)delta g)) with the Pyd substitutes, pyridoxamine dihydrochloride and vitamin B(6), which share the same chemical structure and spectral properties of Pyd cross-links. Neither H(2)O(2) (125-500 microm) nor O(2)((1)delta g) (10-25 microm) significantly changed the spectral properties of pyridoxamine or vitamin B(6). Reaction of HOCl/OCl(-) (12.5-50 microm) with pyridoxamine at pH 7.2 resulted in a concentration-dependent appearance of two new absorbance peaks and a decrease in fluorescence at 400 nm (excitation 325 nm). The new absorbance peaks correlated with the formation of an N-chloramine and the product of its subsequent reaction with pyridoxamine. In contrast, the extent to which HOCl reacted with vitamin B(6), which lacks a primary amine group, was variable at this pH. At lysosomal pH 5.5, Cl(2)/HOCl/OCl(-) reacted with both pyridoxamine and vitamin B(6). Four of the chlorinated products of this reaction were identified by gas chromatography-mass spectrometry and included 3-chloropyridinium, an aldehyde, and several chlorinated products with disrupted rings. To evaluate the effects of Cl(2)/HOCl/OCl(-) on Pyd cross-links in collagen, we exposed bone collagen type I and articular cartilage type II to HOCl. Treatment of either collagen type with HOCl at pH 5. 0 or 7.2 resulted in the oxidation of amine groups and, for collagen type II, the specific decrease in Pyd cross-link fluorescence, suggesting that during inflammation both oxidations may be used by neutrophils and monocytes to promote the loss of matrix integrity.     

Reduced collagen and ascorbic acid concentrations and increased proteolytic susceptibility with prelabor fetal membrane rupture in women

Prelabor rupture of the fetal membranes affects approximately 10% of women at term, resulting in an increased risk of maternal and neonatal infection. Evidence suggests that membrane rupture is related to biochemical processes involving the extracellular matrix of the membranes. We tested the hypothesis that prelabor ruptured membranes are characterized by reduced collagen concentrations, altered collagen cross-link profiles, and increased concentrations of biomarkers of oxidative damage. We also set out to determine whether these effects are modulated by ascorbic acid status. In a case-control study, we explored the role that ascorbic acid, oxidative stress, collagen, and collagen cross-links play in determining membrane integrity and developed a functional assay to assess membrane proteolytic susceptibility. Prelabor ruptured membrane had a reduced ascorbic acid concentration in comparison with controls while protein carbonyl and malondialdehyde concentrations were increased. Collagen concentrations were also reduced in prelabor ruptured membrane, and while the concentration of collagen cross-links was not significantly different between prelabor and timely ruptured membrane, there was a regional variation in cross-link ratio within the amniotic sac. Proteolytic resistance in vitro was reduced in prelabor ruptured membrane and also exhibited regional variation within the amniotic sac. Our findings are strongly supportive of a role for the enhanced degradation of membrane collagen in the determination of prelabor rupture of fetal membranes. The formation of the rupture initiation site is a function of a regional variation in collagen cross-link ratio. Tissue ascorbic acid status may be an important mediator of these processes.