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• W2012328023 abstract "Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen. Matrix vesicles are lipid bilayer-enclosed structures that initiate extracellular mineral formation. Little attention has been given to how newly formed mineral interacts with the lipid constituents and then emerges from the lumen. To explore whether specific lipids bind to the incipient mineral and if breakdown of the membrane is involved, we analyzed changes in lipid composition and extractability during vesicle-induced calcification. Isolated matrix vesicles were incubated in synthetic cartilage lymph to induce mineral formation. At various times, samples of the lipids were taken for analysis, extracted both before and after demineralization to remove deposited mineral. Phosphatidylserine and phosphatidylinositol both rapidly disappeared from extracts made before decalcification, indicating rapid degradation. However, extracts made after demineralization revealed that phosphatidylserine had become complexed with newly forming mineral. Concomitantly, its levels actually increased, apparently by base-exchange with phosphatidylethanolamine. Though partially complexed with the mineral, phosphatidylinositol was nevertheless rapidly broken down. Sphingomyelin and phosphatidylethanolamine also underwent rapid breakdown, but phosphatidylcholine was degraded more slowly, all accompanied by a buildup of free fatty acids. The data indicate that phosphatidylserine forms complexes that accompany mineral formation, while degradation of other membrane phospholipids apparently enables egress of crystalline mineral from the vesicle lumen. Matrix vesicles (MV) 1The abbreviations used are:MVmatrix vesiclesPSphosphatidylserineTEMtransmission electron microscopyHPTLChigh performance thin-layer chromatographyHPLChigh performance liquid chromatographyELSevaporative light scatteringFFAfree fatty acidsMGmonoacylglycerolsCRMVcollagenase-released MVSCLsynthetic cartilage lymphPIphosphatidylinositolCHfree cholesterolDGdiacylglycerolsPEphosphatidylethanolaminePCphosphatidylcholineSPHsphingomyelinLPSlysophosphatidylserineLPIlysophosphatidylinositolLPElysophosphatidylethanolamineLPClysophosphatidylcholine 1The abbreviations used are:MVmatrix vesiclesPSphosphatidylserineTEMtransmission electron microscopyHPTLChigh performance thin-layer chromatographyHPLChigh performance liquid chromatographyELSevaporative light scatteringFFAfree fatty acidsMGmonoacylglycerolsCRMVcollagenase-released MVSCLsynthetic cartilage lymphPIphosphatidylinositolCHfree cholesterolDGdiacylglycerolsPEphosphatidylethanolaminePCphosphatidylcholineSPHsphingomyelinLPSlysophosphatidylserineLPIlysophosphatidylinositolLPElysophosphatidylethanolamineLPClysophosphatidylcholine are extracellular microstructures released by calcifying cells that initiate mineral formation in newly forming bone (1Anderson H.C. J. Cell Biol. 1967; 35: 81-101Crossref PubMed Scopus (282) Google Scholar, 2Bonucci E. J. Ultrastruct. Res. 1967; 20: 33-50Crossref PubMed Scopus (476) Google Scholar, 3Ali S.Y. Sajdera S.W. Anderson H.D. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1513-1520Crossref PubMed Scopus (432) Google Scholar, 4Ianotti J.P. Naidu S. Noguchi Y. Hunt R.M. Brighton C.T. Clin. Orthop. Relat. Res. 1994; 306: 222-229Google Scholar). MV are enclosed by a lipid bilayer membrane that is enriched in selected phospholipids, especially phosphatidylserine (PS) (5Peress N.S. Anderson H.C. Sajdera S.W. Calcif. Tissue Res. 1974; 14: 275-282Crossref PubMed Scopus (130) Google Scholar, 6Wuthier R.E. Biochim. Biophys. Acta. 1975; 409: 128-143Crossref PubMed Scopus (165) Google Scholar), a lipid with known high affinity for Ca2+ (7Nash H.A. Tobias J.M. Proc. Nat. Acad. Sci. U. S. 1964; 51: 476-480Crossref PubMed Scopus (30) Google Scholar, 8Abramson M.B. Katzman R. Gregor H.P. J. Biol. Chem. 1964; 239: 70-76Abstract Full Text PDF PubMed Google Scholar). Initially, PS is largely confined to the inner leaflet of the MV membrane (9Majeska R.J. Wuthier R.E. Calcif. Tissue Res. 1979; 27: 41-46Crossref Scopus (31) Google Scholar). Previous transmission electron microscopic (TEM) studies have shown that the first mineral formed in MV is associated with the inner aspect of the membrane (10Anderson H.C. Bone Miner. 1992; 17: 107-112Abstract Full Text PDF PubMed Scopus (4) Google Scholar). Later, the crystals appear to emerge through the membrane and trigger formation of radial clusters of mineral centered on the remnant of the original vesicle. How the crystals penetrate the MV membrane is currently unknown. While it is possible that simple physical force mediates this process (11Skrtic D. Eanes E.D. Calcif. Tissue Int. 1992; 50: 253-260Crossref PubMed Scopus (15) Google Scholar), there is indirect evidence that latent lipolytic enzymes become activated during Ca2+accumulation and facilitate breakdown of the membrane. To explore this latter possibility, the composition of lipids in MV was analyzed during the course of MV-mediated mineralization in vitro. matrix vesicles phosphatidylserine transmission electron microscopy high performance thin-layer chromatography high performance liquid chromatography evaporative light scattering free fatty acids monoacylglycerols collagenase-released MV synthetic cartilage lymph phosphatidylinositol free cholesterol diacylglycerols phosphatidylethanolamine phosphatidylcholine sphingomyelin lysophosphatidylserine lysophosphatidylinositol lysophosphatidylethanolamine lysophosphatidylcholine matrix vesicles phosphatidylserine transmission electron microscopy high performance thin-layer chromatography high performance liquid chromatography evaporative light scattering free fatty acids monoacylglycerols collagenase-released MV synthetic cartilage lymph phosphatidylinositol free cholesterol diacylglycerols phosphatidylethanolamine phosphatidylcholine sphingomyelin lysophosphatidylserine lysophosphatidylinositol lysophosphatidylethanolamine lysophosphatidylcholine Since previous work had shown that not all lipids were readily extracted from mineralizing tissues (12Eisenberg E. Wuthier R.E. Frank R.B. Irving J.T. Calcif. Tissue Res. 1970; 6: 32-48Crossref PubMed Scopus (29) Google Scholar, 13Wuthier R.E. Gore S.T. Calcif. Tissue Res. 1977; 24: 163-171Crossref PubMed Scopus (117) Google Scholar), resident MV lipids were extracted both before and after demineralization using both neutral and acidic lipid solvents. Lipid composition was analyzed qualitatively by high performance thin layer chromatography (HPTLC) and quantitatively by high performance liquid chromatography (HPLC) using an evaporative light scattering (ELS) detector, which enabled accurate quantitation of the various lipids. Our findings reveal that extensive phospholipid degradation occurred during MV calcification, and this was accompanied by a concomitant rise in the amount of free fatty acids (FFA) apparently released by phospholipases present in the vesicles. The breakdown of MV phospholipids was accompanied by a substantial reduction in the extractability of certain phospholipids, and the composition of MV lipids changed significantly during the process of mineralization. In particular, PS, which became progressively more tightly complexed with nascent mineral, could only be fully extracted after demineralization. It was not only protected from degradation, but was actually synthesized, apparently by a base-exchange mechanism. Large batches of collagenase-released matrix vesicles (CRMV) were isolated from the metatarsal growth plate cartilage of 6− to 8-week-old broiler strain chickens using previously described methods (14Wu L.N.Y. Genge B.R. Dunkelberger D.G. LeGeros R.Z. Concannon B. Wuthier R.E. J. Biol. Chem. 1997; 272: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In brief, cartilage shavings from ∼300 chicken feet (∼160 g) were digested with 0.1% trypsin (type III, Sigma) at 37 °C for 30 min in a synthetic cartilage lymph (SCL) (14Wu L.N.Y. Genge B.R. Dunkelberger D.G. LeGeros R.Z. Concannon B. Wuthier R.E. J. Biol. Chem. 1997; 272: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) with an ionic composition similar to that found to be present in native cartilage (15Wuthier R.E. Calcif. Tissue Res. 1977; 23: 125-133Crossref PubMed Scopus (119) Google Scholar). The trypsin solution was removed; tissue slices were rinsed twice with SCL and digested with collagenase (200 units/g of tissue, type IA, Sigma) at 37 °C for 3−3.5 h. The partially digested tissue was vortexed, and the suspension centrifuged as previously reported to sediment the CRMV (14Wu L.N.Y. Genge B.R. Dunkelberger D.G. LeGeros R.Z. Concannon B. Wuthier R.E. J. Biol. Chem. 1997; 272: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). The pellet was resuspended as a stock containing 5.0 mg of vesicle protein/ml in SCL modified to have one-half the normal level of Ca2+ to prevent dissolution of labile Ca2+ and Pi (16McLean F.M. Keller P.J. Genge B.R. Walters S.A. Wuthier R.E. J. Biol. Chem. 1987; 262: 10481-10488Abstract Full Text PDF PubMed Google Scholar) and also to minimize Ca2+ uptake by the CRMV during storage. Protein levels were determined by the Lowryet al. method (17Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). To have sufficient material for accurate lipid analyses, the large-scale preparations of CRMV (∼20 mg of protein) were allowed to mineralize by incubation in 600 ml of SCL at 37 °C. At each time point (0, 2, 4, 6, and 24 h) for lipid analysis a 110-ml sample was centrifuged at 100,000 ×g for 60 min to sediment the CRMV. The resulting pellets were transferred to individual glass tubes for lipid extraction after resuspension in ∼150 μl of SCL. Mineral formation in samples of the incubation mixture was monitored by light scattering, essentially as described previously (14Wu L.N.Y. Genge B.R. Dunkelberger D.G. LeGeros R.Z. Concannon B. Wuthier R.E. J. Biol. Chem. 1997; 272: 4404-4411Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Lipids were extracted from the CRMV pellets, essentially as previously described (18Wuthier R.E. J. Lipid Res. 1968; 9: 68-78Abstract Full Text PDF PubMed Google Scholar). For qualitative HPTLC analyses, the vesicle pellets were extracted with chloroform, methanol (2:1) (v/v) (∼20 ml/ml aqueous medium), followed by sonication for 1−2 min (extract 1). The tubes were then centrifuged at 3,000 rpm for 12 min to sediment the insoluble residue. Initially, after collecting the lipid-containing supernatant, a second extraction with chloroform,methanol,HCl (200:100:1) (v/v/v) was performed, assuming that any mineral-complexed would be readily extractable. However, subsequent work revealed that significant amounts of the acidic phospholipids remained in the residue. Therefore, for all studies reported here, after the initial lipid extraction, the CRMV pellets were then demineralized with 0.5 m sodium salt EDTA for 20 min at room temperature and sedimented by centrifugation at 3,000 rpm for 12 min. After removal of the supernatant, the decalcified residue was reextracted using chloroform,methanol,HCl (200:100:1) (v/v/v) (extract 2), which was found to quantitatively remove the remaining lipids. The crude extracts were dried under N2 and partitioned through a Sephadex G-25 column to remove non-lipid contaminants (19Wuthier R.E. J. Lipid Res. 1966; 7: 558-561Abstract Full Text PDF PubMed Google Scholar). For qualitative analysis, the pure lipids from the preceding steps were analyzed by HPTLC on Whatman LHP-K silica gel plates as described previously (20Banerjee P. Buse J.T. Dawson G. Biochim. Biophys. Acta. 1990; 1044: 305-314Crossref PubMed Scopus (32) Google Scholar, 21Wu L.N.Y. Yoshimori T. Genge B.R. Sauer G.R. Kirsch T. Ishikawa Y. Wuthier R.E. J. Biol. Chem. 1993; 268: 25084-25094Abstract Full Text PDF PubMed Google Scholar). Mixtures containing 10 μg each of various lipid standards were applied to separate lanes on the same plate as the MV lipid samples. Lipids were visualized by spraying with cupric-phosphoric acid charring reagent (10% CuSO4 in 8% H3PO4) and heating at 180 °C for 10 min in an oven (20Banerjee P. Buse J.T. Dawson G. Biochim. Biophys. Acta. 1990; 1044: 305-314Crossref PubMed Scopus (32) Google Scholar). HTLC plates were photographed and digitized for semi-quantitative estimation of lipid levels. For accurate quantitative analysis, the lipids were analyzed using a Shimadzu HPLC (SCL-10A Systems Controller, SIL-10A Auto Injector, Dual LC-10AT Liquid Chromatographs to provide the gradients). The HPLC system was equipped with an Alltech Varex MKIII ELS detector system. Lipids were separated on a Lichrosorb SI-100 4.6 × 250 mm, 10 μm particle size, HPLC column supplied by Alltech Inc. Injection volume was 30−50 μl (autosampler). The two mobile phases used were as follows: A, methanol,water (80:20) (v/v); B, chloroform,methanol,0.1% formic acid (80:20:0.1) (v/v/v). The following gradient program was used: 1) 92% B,8% A for 6 min; 2) 92% B,8% A to 56% B,44% A in 21 min; 3) 56% B,44% A to 20% B,80% A in 2 min, 4) 20% B,80% A to 100% B in 1 min, 5) 100% B to 92% B,8% A in 3 min. Total run time was 33 min, with a solvent flow rate of 1 ml/min. Nitrogen gas, 11.6 pounds per square inch, was used as the carrier at a flow rate of 2.02 standard liters per min in the ELS detector, which was operated at 72 °C. Mineralization induced by the isolated CRMV was monitored by formation of mineral from the SCL during the incubation period. As had been previously observed, there was a progressive induction of mineral formation during incubation of the CRMV with SCL (Fig. 1 A). As is typical of MV mineralization, there was a short lag period (∼1 h) during which minimal Ca2+ accumulation occurred; thereafter, the rate of mineral formation increased rapidly and by 6 h mineral formation was ∼80% of maximum. Fig. 1 Bshows an electron micrograph of early MV mineralization made after 2 h of incubation in SCL. Three different methods of analysis were used to assess the lipid composition of CRMV, both before and after incubation with SCL, which induced mineral formation. Initially, for qualitative analysis, HPTLC was used to analyze the various polar and nonpolar lipid classes present in extracts of CRMV made before and after demineralization. From these studies it was evident that significant changes in lipid composition were occurring during MV calcification. Most notable was the dramatic disappearance of PS and phosphatidyl inositol (PI) in extracts made before decalcification after only ∼2 h of incubation (Fig.2 A). Also there were progressive increases in the levels of FFA and in the band that included free cholesterol (CH) and 1,2-diacylglycerols (DG). On the other hand, there was a strikingly different lipid composition in extracts made after decalcification (Fig. 2 B). Here there was a dramatic increase in the levels of PS at time points when MV calcification was in rapid progress. While densitometric scans of the HPTLC plates afforded reasonable estimation of the levels of individual lipids present, we used HPLC to more accurately measure the quantitative changes in lipid composition. Initially, we employed a highly sensitive UV detection system at 205 nm to quantitate lipids present (22Hurst W.J. Martin R.A., Jr. J. Am. Oil Chem. Soc. 1984; 61: 1462-1463Crossref Scopus (35) Google Scholar). But because of the variability in sensitivity to individual lipids, which depended on the degree of fatty acid unsaturation, we found this HPLC technique of limited utility. With careful calibration, some useful information was obtained. However, ELS detection proved to be the method of choice for quantitation of the individual lipid classes, since it was not dependent on chromophores. Shown in TableI are the percentages of the total phospholipid of various lipid classes in combined extracts made before and after decalcification at successive time points fromT 0 to T 24.Table IChanges in matrix vesicle lipid composition during in vitro calcificationLipidIncubation timeh024624% of the total phospholipidPE(16) 16.9 ± 1.3(9) 15.8 ± 1.7(9) 12.4 ± 1.1(9) 13.3 ± 1.9(8) 15.7 ± 3.6LPE(16) 6.0 ± 1.0(9) 6.2 ± 1.5(9) 6.1 ± 1.5(9) 6.6 ± 1.8(8) 10.8 ± 2.3PS(16) 9.2 ± 1.7(9) 10.8 ± 2.9(9) 12.6 ± 3.1(9) 15.0 ± 4.3(8) 19.2 ± 4.5LPS(13) 0.05 ± 0.04(6) 0.8 ± 0.5(6) 2.4 ± 1.1(6) 1.5 ± 0.6(5) 3.3 ± 1.8PC(16) 52.1 ± 2.8(9) 57.6 ± 5.9(9) 57.6 ± 6.8(9) 56.1 ± 8.3(8) 38.0 ± 8.3LPC(13) 1.2 ± 0.6(6) 0.6 ± 0.5(6) 0.9 ± 0.6(6) 0.7 ± 0.4(5) 2.7 ± 1.8PI(13) 4.0 ± 0.6(6) 2.8 ± 0.7(6) 2.1 ± 0.9(6) 1.9 ± 0.8(5) 2.0 ± 2.0LPI(12) 1.4 ± 0.4(6) 0.1 ± 0.0(6) 0.2 ± 0.1(6) 0.2 ± 0.2(5) 1.0 ± 1.0SPH(13) 8.4 ± 0.8(6) 5.1 ± 0.7(6) 5.0 ± 1.2(6) 4.2 ± 1.2(5) 2.6 ± 1.4Values are the mean ± S.E. of replicate analyses, the number shown in parentheses before each. These are the sum of extracts 1 and 2 made before and after demineralization, respectively, and are the composite of data from both HPLC and HPTLC analyses of the lipids. The reason there are more replicates shown here than in studies where lipid composition was analyzed separately in extracts made before and after demineralization is because in a number of initial experiments the lipids present in the two extracts were combined before analysis. Open table in a new tab Values are the mean ± S.E. of replicate analyses, the number shown in parentheses before each. These are the sum of extracts 1 and 2 made before and after demineralization, respectively, and are the composite of data from both HPLC and HPTLC analyses of the lipids. The reason there are more replicates shown here than in studies where lipid composition was analyzed separately in extracts made before and after demineralization is because in a number of initial experiments the lipids present in the two extracts were combined before analysis. In general agreement with previous analyses (23Wuthier R.E. Fed. Proc. 1976; 35: 117-121PubMed Google Scholar, 24Wuthier R.E. Wians F.H., Jr. Giancola M.S. Dragic S.S. Biochemistry. 1978; 17: 1431-1436Crossref PubMed Scopus (20) Google Scholar), atT 0, phosphatidylcholine (PC) was the major phospholipid in MV, representing about 50% of the total. Phosphatidylethanolamine (PE), ∼17%, PS ∼9%, sphingomyelin (SPH), ∼8%, monoacyl (i.e. lyso)phosphatidylethanolamine (LPE), ∼6%, and PI, ∼4% were the principal lipids. Low levels of lysophosphatidylinositol (LPI), 1.4%, lysophosphatidylcholine (LPC), 1.2%, and lysophosphatidylserine (LPS), 0.05% were the only other phospholipids consistently detected. With time, in each successive set of extracts, the lipid composition progressively changed. The most notable changes were the significant increases in the levels of PS, LPS, and LPE and the progressive disappearance of both PI and SPH (Table I). However, these values were based on the analyses of lipids present in extracts taken at the successive points during MV calcification. During this period, changes in lipid composition were occurring due to both variable lipid degradation and to specific effects on extractability caused by the selective binding of certain lipids to the newly forming mineral. Because of this, an attempt was made to normalize the progressive changes occurring in total lipid composition by combining data on total polar and nonpolar lipids from extracts taken before and after decalcification at T 0 to serve as a baseline for changes in individual lipid classes occurring at subsequent time points. These changes were then expressed as a percentage of the original amount of each lipid class at successive time points. Combining data on lipids extractable before and after demineralization at each time point caused only modest (±10%) overall change in the total lipid (phospholipid + nonpolar lipid) content to occur during MV-induced calcification. However, when the fate of the total phospholipid was separated from that of the total nonpolar lipid, major progressive changes in each type of lipid were evident (Fig.3 A). Whereas the amount of total phospholipid underwent rapid and progressive decline, the opposite occurred with the nonpolar lipid. After 6 h of incubation, only about 50% of the original phospholipid remained, whereas a nearly 33% increase over the original amount of nonpolar lipid was seen. By 24 h, only about 20% of the total phospholipid remained, whereas the amount of nonpolar lipid had increased 66% above its original level. Examination of the combined data on individual lipids extractable before or after demineralization revealed major differences in the stability of each class of phospholipid (TableII). These revealed that rapid degradation of the acidic phospholipids PS and PI occurred; for both lipids only about 50% of the original remained after 2 h of incubation. However, surprisingly, PS “recovered” and returned to nearly 75% of the original by 24 h. In contrast, levels of PI continued their rapid decline and by 24 h, less than 10% of the original lipid remained. SPH also showed very rapid and continuous degradation, whereas breakdown of PE and PC was significantly slower. The changes in the content of lysophospholipids were complex and will be elaborated on later. However, overall the HPLC findings corroborate and clarify the qualitative impression observed upon HPTLC analyses.Table IIRecovery of matrix vesicle lipids during in vitro calcificationLipidIncubation timeh024624% of the original phospholipid classPE(6) 100 ± 0(6) 84 ± 17(6) 61 ± 11(6) 46 ± 8(6) 25 ± 4LPE(6) 100 ± 0(6) 62 ± 22(6) 55 ± 21(6) 53 ± 25(6) 51 ± 30PS(6) 100 ± 0(6) 56 ± 14(6) 72 ± 8(6) 73 ± 9(6) 77 ± 12LPS(3) 100 ± 0(3) 108 ± 10(3) 193 ± 12(3) 119 ± 13(3) 199 ± 56PC(6) 100 ± 0(6) 87 ± 16(6) 79 ± 16(6) 70 ± 15(6) 24 ± 11PI(6) 100 ± 0(6) 48 ± 20(6) 19 ± 10(6) 16 ± 10(6) 8 ± 8SPH(6) 100 ± 0(6) 63 ± 15(6) 34 ± 11(6) 22 ± 7(5) 7 ± 5Total(6) 100 ± 0(6) 70 ± 12(6) 61 ± 1(6) 52 ± 9(5) 22 ± 4Values are the mean ± S.E. of replicate analyses, the number shown in parentheses before each. These are combined data from extract 1 plus extract 2 made before and then after demineralization, respectively. Values are a composite of data from both HPLC and HPTLC analyses of the lipids. Open table in a new tab Values are the mean ± S.E. of replicate analyses, the number shown in parentheses before each. These are combined data from extract 1 plus extract 2 made before and then after demineralization, respectively. Values are a composite of data from both HPLC and HPTLC analyses of the lipids. As MV calcification progressed with time, the percentage of the total phospholipid extractable before demineralization decreased concomitantly, and correspondingly, the proportion extractable only after demineralization increased (Fig. 3 B). At T 0, nearly 95% of the total phospholipid present was extractable before demineralization; however, after 4 h of incubation, by which time mineralization was well in progress, less than 80% was extractable. By 24 h, only about 70% of the remaining phospholipid could be extracted prior to demineralization. This indicated that with calcification, progressively more of the surviving phospholipid was bound to the mineral and could not be extracted until the vesicles were demineralized. In contrast, the extractability of the nonpolar lipid was much less affected by mineralization. At T 0, about 90% of the nonpolar lipid was extractable before demineralization. With time this increased to about 93–94% (data not shown). Corroborating the impression gained by HPTLC, analysis by HPLC revealed that MV mineralization had a profound effect on the extractability and survival of the two principal acidic phospholipids, PS and PI. At T 0 before exposure to the mineralizing solution, about 90% of the PS was extractable before demineralization; however, after only 2 h of incubation, only 5% of the original PS was extractable (Fig.4 A). There was a corresponding major increase in the proportion of the original PS that survived and could be extracted only after demineralization. This increased from ∼10% at T 0 to about 75% atT 4 and remained at this level thereafter.Figure 4Changes in the percentage of individual phospholipid classes recoverable in extracts made before (closed symbols) and after (open symbols) demineralization. A, phosphatidylserine (PS), B, phosphatidylinositol (PI),C, phosphatidylethanolamine (PE), D,phosphatidylcholine (PC), E, sphingomyelin (SPH), F, lysophosphatidylethanolamine (LPE), and G, lysophosphatidylserine (LPS). Data presented are the percentages at each time point during progressive stages of MV calcification of the original amount of each phospholipid class recovered in extracts made before and after demineralization, expressed as the mean ± S.E. The means are from six separate analyses for all lipid classes except SPH, for which only five analyses were available at 24 h and LPS, for which only three analyses were available at all time points.View Large Image Figure ViewerDownload (PPT) A similar pattern of extractability was seen with PI, but as noted in Table II, there was major degradation of this lipid during MV mineralization. At T 0, again about 90% of the PI was extractable, but after 2 h of incubation, in contrast to PS, almost one-third of the original PI could still be extracted (Fig.4 B). However, by T 4 and thereafter, no PI was extractable before demineralization. Again, in contrast to PS, there was much less of the original PI that could be extracted after demineralization. This value rose from ∼7% atT 0 to a maximum of only about 18–20% of the original at T 4, decreasing gradually thereafter. This indicated that much less of the original PI became complexed and stabilized by the newly forming mineral and thus was susceptible to degradation. In contrast to the acidic phospholipids, mineralization had only a minor effect on the extractability of the neutral phospholipids. For PE, about 90% of the original was extractable before demineralization at T 0. The remaining 10% was apparently complexed with the mineral and not extractable (Fig. 4 C) and was protected from degradation, persisting throughout the 24 h of incubation. However, the amount of PE that was not complexed rapidly decreased, indicating that it was rapidly degraded. Essentially all (>95%) of the choline-containing phospholipids, PC and SPH, were extractable before demineralization, regardless of the length of incubation or extent of mineralization. Fig. 4 Dreveals that PC was readily extractable before demineralization; however, its rate of degradation was slower than most other phospholipids, remaining essentially linear throughout the incubation. By 24 h, only about 20% of the original PC remained. Fig.4 E shows that SPH, also unprotected by complexation with the mineral, was rapidly degraded. By 4 h, only about one-third of the original SPH remained, and by 24 h there was only about 5% of the original left. Thus, in contrast to all of the other diacyl phospholipids, PS largely survived being protected by the newly forming mineral and resynthesized from other lipids (see later). Evident in all lipid extracts of MV were significant amounts of lysophospholipids. LPE, the monoacyl derivative of PE, was the most abundant form representing ∼6% of the total phospholipid for most of the incubation (Table I). As evident from Fig. 4 F, almost 95% of the LPE present initially was extractable before demineralization. However this rapidly declined; by 4 h only about one-third, and by 24 h less than 20% of the original LPE could be so extracted. On the other hand, the amount of LPE not extractable until after demineralization increased progressively during MV calcification, rising from ∼5% atT 0 to ∼35% at T 24. The other lyso-derivatives detected during MV calcification were LPI, LPC, and LPS. Small amounts of LPI and LPC (1.4 and 1.2% of the total PL, respectively) were present initially, but there were only trace amounts of LPS (Table I). While levels of LPI and LPC generally decreased thereafter, levels of LPS increased. At 4 h, in lipids extractable after decalcification, levels of LPS were dramatically increased (Fig.4 G). This corresponds to the ti" @default.
• W2012328023 created "2016-06-24" @default.
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• W2012328023 date "2002-02-01" @default.
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• W2012328023 title "Changes in Phospholipid Extractability and Composition Accompany Mineralization of Chicken Growth Plate Cartilage Matrix Vesicles" @default.
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