The Nature of Phospholipids in Normal and Rachitic Costochondral Plates By DAVID S. HOWELL, JUAN F. MAHQUEZ AND Interest in fundamental biochemical processes ol mineralization in osteoid led to a study of rachitic calf costal junctions in respect to a profile of phospholipids. Currently, chloroformmethanol extracts of rachitic and normal costochondral junctions were applied to thin-layer silica gel chromatograms, and the phospholipids thereby separated were quantitated in terms of phosphorus content of the eluted spots. Striking differences between normal and rachitic tissues were found in respect to this profile of phospholipids. JULIO C. PITA Rachitic junctiones costal de vitello esseva studiate relative a lor profilo phospholotidic. Le studio esseva interprendite conio contribution a1 investigation del basic processos biochimic participante in le mineralisation de osteoide. Extractos a chloroformo e methanol ex normal e rachitic junctiones costochondral esseva applicate a systemas chromatographic a gel de d i c e in strato tenue, e le assi-separate phospholipidos esseva quantificate relative a1 contento de phosphor0 del areas eluite. Frappante differentias esseva constatate inter tissues normal e tissus rachitic. A CONSISTENT SELECTIVE STAINING of mineralizing sites in a variety of tissues by Sudan black'J was demonstrated by J. T. Irving and co-workers.' Histochemical studies of extracellular sites led to a tentative identification of the material as a lipide2 Sudan black as well as the Baker's stain for phospholipids and postulated a distinctive role for phospholipids in formation of mineralizing matrix.3 In a prior study of Ca4?uptake by substances in aortic walls in vitro, Fels surmised calcium binding by a phospholipid-protein c ~ m p l e x .The ~ fact that the Sudan black staining reactions for lipids were not noted in dentinal pulp or rapidly growing periosteal membranes directly adjacent to the sites of mineral deposition, or in rachitic osteoid, suggested that this staining is not a property of a nonspecific proliferating fibrous matrix but rather related distinctively to those extracellular sites involved in the process of mineral up take. ' , ~ 2 0 A regulatory function for lipids in cell membrane transport of electrolytes and other substances has received considerable attention, as recently reviewed," so that a possible selective role for these compounds in mineral ion or water transfer deserves exploration at calcifying regions.5~6~17~'s~24 The present report concerns quantitative identification of one class of lipids in relation to histologic sites of normal mineralization in contrast to From the Division of Arthritis, Department of A4 edicine, University of Miami S c b o l of Medicine, Miami, Flu. Supported by Grants AM 08662-01 and A-1155 of the National Institutes of Arthritis und Metabolic Diseases, Bethesda, Md. DAVID s. HOWELL,M.D.: Associate Professor of Medicine; JUAN I?. MAHQUFZ, M.D.: Reser~rclaInstructor; JULIO C. PITA, P H . ~: .Visiting Professor of Physical Chemistry; a11 of the Depnrtmcnt of Medicine, University of Minmi School of Medicine, University of M u i m i , Miami, Flu. 1039 1040 HOWELL, MARQUE2 AND PITA similar poorly mineralized sites of rachitic tissues. Rachitic calf costal and adjacent costal osteoid was analyzed for phospholipid content and the results compared to a similar assessment of normal juxtametaphyseal cartilage and osteoid." These preliminary observations indicate that, in addition to the welldocumented electrolytic abnormalities of nutritional rickets, there are probably important differences in phospholipid profile between a rachitic and normal cartilage and osteoid. MATERIALSAND METHODS I. Tissue Preparation and Histologic Description of Tissue Fractions Rachitic calves were prepared at the Cattle Breeding Institute, Wiad, Sweden, as described elsewhere.? Normal (control) calves were raised in a similar environment and received a similar diet with the exception that whole milk, hay, supplementary mixtures of vitamin D, calcium, and phosphorus were added. The experimental animals developed weakness, ataxia, lameness and excessive epiphyseal prominences of their extremities as predicted from previous studies.8,g Blood samples from 3 of the rachitic calves, obtained at the time of sacrifice, revealed a low serum (calcium) x (phosphate) concentration product in each instance. Three calves were sacrificed 1-3 weeks after the first objective clinical manifestations of rickets, which appeared at 4 to 6 months of age. A fourth animal died from an unidentified infection, and some of the rachitic tissues showed spontaneous healing. Four control animals developed satisfactorily and were sacrificed concurrently with the experimental group. Following sacrifice the costochondral tissues of the first 9 ribs, bilaterally, were dissected free and quick-frozen in isopentane surrounded by liquid nitrogen. The costochondral junctions from rachitic, as well as normal calves, were cleaned of surrounding looye connective tissues, divided sagitally and cross-sectioned into 3 fractions under a dissecting microscope. These fractions included predominantly: ( 1) Resting and proliferating cell cartilage (fractions A-I and B-I); ( 2 ) hypertrophic cell cartilage and adjacent coatings of osteoid (fractions A-I1 and B-II), and ( 3 ) metaphyseal osteoid (fraction B-111). Fraction A-I11 contained bone marrow, principally of the cellular variety, and trabecular bone, but was not analyzed biochemically for the present study. A description of histologic methods and tissue separation has been given in detail previously.7-15 Representative samples of these tissue regions, studied biochemically, were sectioned, frozen in a cryostat, and stained with hematoxylin and eosin, as well as silver nitrate, by the Von Kossa method to assess mineral distribution. Sudan black was also employed, including the pyridine extraction step of Irving.1 2. Biochemical Methods Representative samples of each fraction were diced and ground to 60 mesh, tared, wet, and dried over calcium chloride in a dessicator to constant weight. The dried tissues were extracted 3 times with cold 0.9 per cent buffered saline to remove water soluble contaminants. Residues were suspended in chloroform and reground in a Virtis-45 homogenizer for 1 hour. Ten ml. samples of the suspension were placed in a separatory funnel and washed with 5 ml. of .02 M sulfuric acid. Three such washings were followed by 2 additional extractions with chloroform-methanol, 2:l mixture. The extracts were pooled and dried by lyophylization. Total phospholipid was cstimated from these residues by total phosphorus content, employing a conventional phosphomolybdic acid procedure.15 For identification of individual phospholipids, ascending silica gel chromatography was used.12 Plates were prepared according to Stah111 and were composed of a neutral thin layer (0.25 mm. thickness) silica1 gel G# 7731, E. Merck AG Darmstadt, Germany, 20 ~ *Some unmineralized juxtacartilagin~usborders justify this term in these control ribs. 1041 NATURE OF PHOSPHOLIPIDS Gm. in 50 ml. of water. Chromatograms were developed as described by Wagner13 for a distance of 12 cm. in chloroform-methanol-water, 65:25:4. The mean Rf for known samples of phosphatidylethanolamine was 4.6 cm. for phosphatidylcholine, 2.4 cm. sphingomyelin, paired spots, 1.9 and 1.6 cm. respectively, and lysophosphatidylcholine 1.1 cm. The points of migration were identified with iodine vapor. Corresponding spots were removed from the plates, digested in perchloric acid-nitric acid, 4:1, according to Bartlettl4 and total phosphorus content measured as mentioned above. Slight modification in procedure and calculation of recoveries of p on chromatogram were made according to Slotta.10 Using purified calf brain phosphatidylethanolamine added to the starting material, 95-97 per cent recoveries were obtained in 4 analyses. This phosphatide was also used to verify the Rf values mentioned above.* Reproducibility of results on duplicate samples and recovery of P in phospholipid analyses of mitochondria were compared with and without purification of starting extracts on chromatographic columns charged with silica gel; since the extra step failed to improve reproducibility of results, the step was omitted.10 Also, partial loss of Sudan black staining in sections, treated briefly with EDTA, interdicted steps to remove mineral from starting samplcs other than through mild hydrolysis. RESULTS Histologic assessments of rachitic tissue fractions B-I and B-11, in respect to cellular composition, indicated similar cell types to those of A-I and A-I1 fractions respectively. The currently studied tissue samples were similar to those analyzed for mucopolysaccharides previously in respect to histochemical evidence of lipid distribution.? Thus, Sudan black staining by the method of Irving1 including the pyridine extraction step, registered slight but consistently positive reactions in all of the cartilage cells and a strongly positive reaction bordering bone salts in the extracellular matrices of fraction A-I1 cartilage and osteoid (fig. 1 ) . All histologic regions of fractions A-I, B-I, and B-I1 were negative with Sudan black staining except at intracellular sites. The number of cells per mm.3 of fixed tissue measured in histologic sections was strikingly similar to that of normal calf plates of the same age, reported previously.15 The current counts, based on 12 fields each from 60 histologic sections, were: 75 5 (SE) for hypertrophic cell zone, 196 3 for proliferating cell zone, and 59 7 for resting cell zone in comparison 1 for respective zones in normal ~ 1 a t e s .Conl~ to 57 +- 1, 199 4,and 66 current cell counts on rachitic osteoid were 109 3. Total nitrogen content per unit dry weight of original normal tissue (A-I), and that of rachitic samples (B-I), lacking mineral salts as seen by histologic examination, were comparable; total nitrogen content of fractions A-I1 and B-I11 was lower, probably because of a large component of bone salts (table 1 ) . Total phospholipid content in the extracts of the rachitic cartilage (fractions B-I, B-11, and B-111) was markedly less than in the extracts of the corresponding regions from normal cartilage (fractions A-I and A-I1 in table 1) In extracts of all the rachitic fractions in comparison to normal controls the proportion of lipids with the Rf value of phosphatidylethanolamine was lower, phosphatidylcholine the same, and sphingomyelin elevated. Conse- * * * * * 'For detection of small amounts of other phosphatides, a two-dimensional paper chromatographic method has proved useful.21 Fig. 1.- ( A ) Zones of provisional calcification and hypertrophic cells (fraction A-11, table 1) revealing extracellular staining at sites of mineral deposition (200x Sudan black). ( B ) Osteoid and cartilage of distal hypertrophic cell areas of a rachitic calf (fraction B-11, table 1) . Only cellular components stained positively ( 2 0 0 ~Sudan black). ( C ) A thin-layer silica gel chromatogram stained with ammonium molybdate-perchloric acid spray. Abbreviations: 0 = Origin; remainder indicate phospholipid bands as described in table 1, (fraction B-11) , ( D ) Metaphyseal bone (fraction N-111) of rachitic calf, also showing wide borders of osteoid. There was no staining in the rachitic osteoid and only weak staining of compact bone (200 x Sudan black). 1043 NATURE OF PHOSPHOLIPIDS quently, in all tissue fractions the ratio of phosphatidylcholine to phosphatidylethanolamine was markedly higher in the presence of rickets. In respect to fractions A-I1 and B-11, the proportion of lipid with the Rf of lysophosphatidylcholine was less in the extracts of rachitic tissue. Only 5-10 per cent of phospholipid phosphorus failed to migrate from the origin in the chromatograms, indicating that if phospholipid-protein complexes were an important fraction of the extract applied, this did not interfere with mobility of the phospholipids. As in a previous report, sulfated lipids were not detected in the rachitic tissues.7 DISCUSSION A difference of cartilage metabolism of phospholipids between animals with nutritional rickets and controls was demonstrated by this study. The uniformly low concentration of phospholipids from the rachitic tissue extracts are compatible with either depressed synthesis or accelerated degradation at these tissue sites, or both. The large difference of phospholipid content between normal and rachitic tissues might have been partially a reflection of difference in cell numbers not registered by the current cell counts, which were confined to fixed tissues-wherein heterogeneity interdicts more than a crude assessment of this parameter. Nevertheless these counts favor a quantitative reduction of phospholipid per unit cell in tissues lacking more than a trace of extracellular lipids histochemically. Whether tissues other than cartilage share in this abnormality was not examined. Although the rachitic calves were force fed and gained weight steadily during their experimental period prior to sacrifice,7 total weights were less than that of control calves; and, by gross inspection, these calves appeared less adipose. Fat content of diets in control and experimental calves was comparable. However, dietary components may influence tissue phospholipid^,^^ and an effect on the metabolism of phospholipid constituents in the experimental group receiving coconut and linseed oil supplements might have occurred.7 This explanation is unlikely, however, because similar differences in phosphatidylcholine and phosphatidylethanolamine content between rachitic and normal rat epiphyses have been observed (unpublished observations) at age 25-30 days with employment of entirely different dietary regimens and fat constituents. It was found by Cipera et a1.I6 that during healing of rickets in chicks there was a substantial increase in the acetone-soluble fraction of epiphyseal cartilage per unit dry weight. Such results during healing are at least consistent with a possible relative deficiency of fatty compounds in the cartilage of rachitic chicks. A definite alteration in phospholipid profile seemed evident here (table 1). Although with the mucopolysaccharides, previously studied on these tissues,7 no specific pattern of reduction of chondroitin sulfate A, C, or kerato sulfate was distinguished, herein differences in respect to the relative content of phosphatidylethanolamine and sphingomyelin were found (table 1) . Hyaline cartilage, which was demonstrated to be uncalcified in terms of the Von Kossa stains, exhibited the same pattern of alterations in respect to these particu- Resting &proliferating cell cartilage Hypertrophic cell cartilage & osteoid Metaphyseal osteoid Resting & proliferating cell cartilage Hypertrophic cell cartilage & osteoid Predominant Histologic Region 6.16 fO.l 9.35 fO.l 10.2s +0.111 9.51 f0.3 11.29 kO.111 % 0.137 +0.008 0.322 k0.007 0.090 t0.004 zko.02 0.715 1.290" zk0.002 Total Phospholipidst mg. TO Rachitic 16.6 16.5 19.3 4 17.8 21.6 4 20.1 33.9 f0.55 4 24.6 34.6 f0.94 32.6 i0.72 30.4 k0.94 33.4 f0.80 'ib Phosphatidylchohe: (PC) i0.60 4 k 1.0 26.1 23.5 k0.84 32.8 22.1 37.5 k1.6 % .a. Ccmtrol 19.8 Phosphatidylethanolaminet (PE) 11.3 4 32.8 28.5 +0.85 28.3 f1.5 18.9 21.3 17.9 k1.2 % Sphingomyelrnl (SPG) Normal and Rachitic Osteoid and Cartilage Recovery of Phospholipid-P in 4 Fractions Columns 5-8 of 17.5 23.0 22.0 MLg. Total Phospholipid-P Applied Phospholiuid Components Total Nitrogen* .- Maior Number samples 6 6 analyzed per fraction *Nitrogen and phospholipids are calculated on basis of dry weight, original tissue. +Phospholipid value = Phosphorus 25. $Percentages in columns 5-8 are referred to total phospholipid-P applied to chromatograms. $Mean. I1S.D. B-I11 B-I1 B-I A-I1 A-I Fraction Number Table 1 k0.62 4 9.43 9.80 f0.56 15.6 f1.1 16.6 f1.0 11.7 zko.90 'ib Lysophosphatidylcholinet (LPC) 1.38 1.32 1.38 0.89 0.89 Ratio PC/PE 3 N El ro G z rr 8 1045 NATURE OF PHOSPHOLIPIDS lar phospholipids as the osteoid fractions, thereby indicating a general relationship to the rachitic state in these connective tissues. The polysaccharide content7 was similarly low within the noncalcified as well as within mineralrich tissue fractions. It is unlikely that traces of blood or plasma lipids contained within the samples importantly influenced current data. Fraction B-I, virtually lacking blood vessels, did not reveal, in regard to the 3 phosphatides already discussed, phospholipid profile different from that of B-11, which contained numerous capillaries. Inasmuch as a large component of the phospholipids of fraction A-I1 were probably extracellular ( fig. I-A), this A-I1 phospholipid profile is believed to approximate most closely that of the extracellular matrix at sites stained by Sudan black. The finding of a high proportion of lysophosphatidylcholine encourages further examination of lipid profiles for unique features which might be amenable to study in model systems of in vitro calcification. In view of the effect of vitamin D uptake on calcium translocation in kidney mitochondria rich in phospholipid^,^^ the selectivity of cation transfer through membranes coated with phospholipid,24 and the histochemical identification of phospholipids at mineralizing sites in bone and dentine,2,3 the role of lipids and particularly phospholipids in mineralization of these tissues demands further scrutiny. SUMMARY 1. Phospholipid content of costal cartilage and osteoid in calves with nutritional rickets was approximately one-tenth to one-half that found in costochondral junctions dissected from normal animals of comparable age. 2. In the rachitic tissue, the proportion of phosphatidylethanolamine was decreased and sphingomyelin increased, whereas that of phosphatidylcholine remained constant when compared to normal. 3. One feature that distinguished normal from rachitic osteoid-cartilage junctional tissue was a higher proportion of phospholipid with the Rf of lysophosphatidylcholine. Possible causes of these differences in phospholipid distribution are discussed. ACKNOWLEDGMENTS The authors are indebted to Professors A. Hansson and A. Neimann-Sorensen as well as to Agronomists L.-E. Liljedahl and T. Henningsson for the use of facilities in the preparation of rachitic calves. REFERENCES 1. Irving, J. T.: Histochemical changes in early stages of calcification. Clin. Orthop. 17:92, 1960. 2. Irving, J. T.: The sudanophilic material at sites of calcification. Arch. Oral Biol. 8:735, 1963. 3. Johnson, L. C.: Mineralization of turkey leg tendon. 1. Histology and histochemistry of mineralization. Calcifica- tion in biological systems. R. F. Sognnaes, Ed. Washington, D. C., Am. Assoc. for the Advancement of Science, 1960, p. 117. 4. Fels, I. G.: Binding of calcium ions by the aorta. Nature 190:1012, 1961. 5 . Burgen, A. S. V.: Symposium on the nature of lipoproteins. The structure and function of cell membranes. 1046 Canad. J. Biochem. Physiol. 40: 1253, 1962. 6. Wooley, D. W., and Campbell, N. K.: Tissue lipids as ion exchangers for cations and the relationship to physiologic processes. Biochim. Biophys. Acta 57:384, 1962. 7. Howell, D. S.: Histologic observations and biochemical composition of rachitic cartilage with special reference to niucopolysaccharides. Arthritis Rheum. 8:337, 1965. 8. Landsburg, K. G., and Hill, 0. J.: Rickets in calves. Penn. Agricultural Experiment Station Tech. Bull. 291, 1963. 9. Huffman, C. F., and Duncan, C. W.: Vitamine D studies in cattle. I. The antirachitic value of hay in the ration of daily cattle. J. Dairy Sci. 18:511, 1953. 10. Slottn, K. H.: Mitochondria1 phospholipids in rats of various ages. J. Geront. 18:236, 1963. 11. Stahl, E.: Thin layer chromatography. IV. Insertion scheme, marginal effect, “Acid and base layers,” gradation technic. Arch. Pharm. ( Weinheim) 292/64:411, 1959. 12. Mangold, H. K.: Thin-layer chromatography of lipids. J. Amer. Oil Chem. Soc. 38:708, 1961. 13. Wagner, H., Horhammer, L., and Wolff, P.: Dunnschichtchromatographie von Phosphatiden and Glykolipiden. Biochem. Z. 7 (334)175, 1961. 14. Bartlett, G. R.: Phosphorus assay in column chromatography. J. Biol. Chem. 234:466, 1959. 15. Howell, D. S., DelchampT, E., Reimer, W., and Kiem, I.: A profile of electrolytes in the cartilaginous plate of HOWELL, MARQUE2 AND PITA growing ribs. J. Clin. Invest. 39:90, 1960. 16. Cipera, J. D., Migicovsky, B. B., and Belanger, L. F.: Composition of epiphyseal cartilage. I. Changes in hexosamine and acetone extractable contents of epiphyseal cartilage of rachitic chicks following administration of Vitamin D3. Canad. J. Biochem. Physiol. 38:807, 1960. 17. Zambotti, V., Cescon, I., Bonferroni, B., and Bolognani, L.: Lipids of epiphyseal cartilage. Experentia 18: 318, 1962. 18. Leach, A. A.: The lipids of ox compact bone. Biochem. J. 69:429, 1958. 19. Leat, W. M. F.: Serum phospholipids of pigs given different amounts of linoleic acid. Biochem. J. 91:437, 1964. 20. Howell, D. S., and Carlson, L.: Sulfur metabolisin in cartilage. A study of calcifying regions for microscopic distribution of sulfur and relationship to staining by Sudan black. Exp. Cell. Res. 34:568, 1964. 21. Wuthier, R. E., and Irving, J. T.: Un published observations. 22. Howell, D. S., Marques, J., and Rydberg, M.: The nature of phospholipids in calcifying epiphyseal plates, Clin. Res. (abst.) 12:90, 1964. 23. De Luca, H. F., Engstrom, G. W., and Rasmussen. H.: The action of Vitamine D and parathyroid hormone in citro on calcium uptake and release by kidney mitochondria. Proc. Nat. Acad. Sci. (U. S. A.) 48:1604, 1962. 24. Mikulecky, D. C., Tobias, J. M.: Phospholipid-cholesterol membrane model. J. Comp. Cell Physiol. 64:151, 1964.