HYDROXYAPATITE PRECIPITATION IN VITRO BY LIPIDS EXTRACTED FROM MAMMALIAN HARD AND SOFT TISSUES A. A. ODUTUGA, R. E. S. PROL T and R. J. HOARE The Department of Biochemistry. University of ShefYield. Sheffield SIO ZTN. England Summary-Lipid was extracted from human teeth, rat teeth, rat liver and rat kidney. Total lipid, total neutral lipid, total phospholipid and individual phospholipids were added to metastable calcium phosphate and calcium carbonate solutions. Apatite and calcite respectively were precipitated except in the presence of neutral lipid when no precipitation occurred. Phospholipids were bound to the precipitated apatite and could only be extracted after demineralization.
Histochemical studies on the changes in the organic matrix of bone, dentine and enamel immediately before mineralization indicate that lipids (in particular phospholipids) may be involved in this process (Irving, 1963, 1965, 1973a; Irving and Wuthier, 1968). Histological staining of lipid in teeth and the extraction, particularly of phospholipid, occurs only after decalcification of the tissue (Allred, 1968; Shapiro, Wuthier and Irving, 1966; Prout, Odutuga and Tring, 1973). in uitro experiments have shown that phospholipids isolated from Bacterionma mtruchotii, a microorganism which forms apatite intracellularly, precipitate hydroxyapatite from a me&table solution of calcium phosphate (Takazoe, Vogel and Ennever, 1970). Cotmore, Nichols and Wuthier (1971) have shown that acidic phospholipids combine with calcium phosphate at pH 7.0 to form a precipitate of calcium phosphate and lipid from which the lipids could not be extracted by organic solvents. The purpose of the present study was to determine whether lipids extracted from hard and soft tissues could cause precipitation of hydroxyapatite from a metastable solution of calcium phosphate and, if so, to determine which particular lipids were responsible and the chemical nature of the precipitate. MATERIALS
AND
METHODS
Enamel-dentine mixtures were obtained from freshly extracted human molars and from the incisors and molars of 3-4-month-old Wistar rats; livers and kidneys were also obtained from the same rats which had been maintained on Oxoid modified diet 86 stock rat cubes (Herbert C. Styles (Bewdley) Ltd., Bewdley. Worcs.). The enamel-dentine samples were obtained and the lipids extracted after demineralization as deTable
I. Composition
Calcium phosphate solution in 0.04 M Verona1 buffer (PH 7.0) Calcium carbonate solution
of metastable
scribed by Prout rt al. (1973) and Prout and Odutuga (1974a). Liver and kidney lipids were extracted and purified by the method of Folch. Lees and SloaneStanley (1957). Neutral lipids and phospholipids from the various tissues were separated as described by Odutuga and Prout (1973). A metastable solution of calcium phosphate was prepared essentially by the method of Takazoe rt ~1. (1970) and of calcium carbonate as described by Bachra. Trautz and Simon (1963) (Table 1). To prevent bacterial contamination 200mg of thymol was added per litre of solution. Weighed quantities of either total lipid, neutral lipid, phospholipid or pure commercial lipids (Sigma Chemical Co., London, England) were added to the metastable solutions in conical flasks which were then sealed with Parafilm and aluminium foil. The mixtures were stirred, forming an emulsion, with magnetic stirrers for 7-21 days at room temperature. they were then centrifuged at 30,OOOg for 20 min at 5’C and any precipitate was collected. Precipitates were washed 4 times with distilled water and 3 times with 50ml of chloroform-~ methanol (2: 1, v/v) to remove lipid. The final residues were washed with ethyl alcohol, dried under nitrogen. weighed and stored at -20°C. Portions of the residues were dissolved in 5 N HCl and aliquots analysed for calcium (Kingsley and Robnet. 1957) and phosphorus (Fiske and Subbarow, 1925); the remaining acid solution was neutralized with NaHCO, and lipids were extracted and analysed qualitatively and quantitatively (Prout et al., 1973). Samples of the lipids extracted from the four different tissues (human enamel--dentine, rat enamel-dentine. liver and kidney) were also analysed (Prout cf al.. 1973; Prout and Odutuga, 1974a, b). X-ray diffraction analysis was carried out on the washed and dried precipitates. Samples were mounted
calcium phosphate
NaCl
KC1
70.0 145.0
4.7 5.0
and calcium carbonate
NaHCO,
22-o 22.0 III
Na2HP04
3-9 O-033
solutions
in mM
CaCI, .2Hz0
1a 75.0
pH
7.0
7.2 + 0.05
312
A. A. Odutuga, R. E. S. Prout and R. J. Hoare
Table 2. Composition
Source of lipid
of apatite
precipitated
No. of experiments
Total lipids Human teeth Rat teeth Rat liver Rat kidney Total phospholipids Human teeth Rat teeth Rat liver Rat kidney
from metastable calcium phosphate from different tissues
Lipid extracted from demineralized precipitate o/;,
solution
by lipids extracted
Ca/P in the precipitate (molar ratio)
Phosphorus in the precipitate
Calcium in the precipitate %
0,
/O
6 6 7 7
1.79 1.48 1.94 2.08
* * f +
0.21 0.28 0.26 026
35.61 35.97 34.96 35.66
+ f f +
064 0.35 0.39 0.24
17.97 f 17.41 i 17.72 + 17.65 f
0.09 0.18 033 0.22
1.54 1.60 1.53 1.57
* F * *
0.04 0.02 0.04 003
3 4 3 3
I.17 1.68 I.86 1.67
+ f + k
0.20 0.14 052 0.29
34.91 36.21 35.39 35.54
k + f +
0.61 0.21 0.45 0.45
17.48 17.27 17.25 17.82
0.22 0.16 0.15 0.19
1.56 1.63 1.59 I.55
+ + + +
0.02 0.02 0.06 0.02
in 0.5 mm quartz capillaries and X-ray patterns were recorded using filtered copper radiation and a flat plate camera. Phospholipids containing [l-‘4C]-linoleic acid were obtained from livers and kidneys of 17 normal 3-4-month-old Wistar rats that had been injected 8 days previously with 750 &i of [l-‘“Cl-linoleic acid. The radioactivity of the phospholipid was determined in disintegrations per minute (dpm) per mg as previously described (Prout and Odutuga, 1974b). Radioactive phospholipids were added to metastable calcium phosphate solution as described above. RESULTS
The total lipids extracted from human teeth, rat teeth, liver and kidneys caused the precipitation of calcium phosphate from the metastable solution. The percentages of calcium and phosphate in the precipitates are shown in Table 2, together with the calciumphosphorus molar ratio. X-ray analysis of these precipitates shows them to be apatite, except in one sample precipitated by rat liver total lipid and one sample precipitated by rat kidney total lipid. The weight of apatite precipitated from the metastable calcium phosphate solution is directly proportional to the amount of total lipid added, up to approximately 150mg lipid/lOOml of solution (Fig. 1). The weight of precipitate is slightly lower when the total lipids extracted from human and rat teeth were used than with total lipids from rat liver and kidney. X0 mg/lOO ml very little further precipitation
In the second experiment, total lipids were extracted from human teeth and rat teeth, liver and kidneys and separated into two fractions, one containing total neutral lipid and free fatty acids and the other total phospholipid. The total phospholipids were added to the metastable calcium phosphate solution, and precipitates formed with the composition shown in Table 2 and which X-ray analysis showed to be apatite. The weight of precipitate was slightly higher with total phospholipids from human and rat teeth than when phospholipids from rat liver and kidney were used to promote precipitation (Fig. 1). The amount of phospholipid incorporated into the precipitated apatite varied between 1.2-1.9 per cent (Table 2), depending on the origin of the phospholipid. As with total lipid, the weight of precipitate was directly
Above
occurs. The apatite crystals precipitated by the total lipids from the four tissues contain lipid, which could not be removed by washing with chloroform-methanol unless the precipitate was dissolved in acid when lipid was released which could be extracted from the solution. The lipid bound to the precipitated apatite is shown in Table 2 as a percentage by weight of the precipitate, the percentage being between 1.5-2.0, depending on the origin of the lipid used to promote precipitation.
k + k f
I 0
-
1
,
50
100
I
I50
1
200
mg lipid Fig. 1. Weight of apatite (including bound lipid) precipitated from 100 ml of metastable calcium phosphate solution by differing amounts of lipid from different sources as follows: A, total phospholipid extracted from rat teeth; a, total phospholipid extracted from human teeth; 0, total phospholipid extracted from rat kidney; n, total phospholipid extracted from rat liver; 0, total lipid extracted from rat liver; 0, total lipid extracted from rat kidney; @, total lipid extracted from rat teeth;m, total lipid extracted from human teeth.
Hydroxyapatite
precipitation
by lipids
Table 3. Precipitation of apatite from 100 ml of metastable calcium phosphate solution using 150 mg of phospholipid in each case.
wt. of precipitate (mg) Lecithin Sphingomyelin Phosphatidyl ethanolamine Phosphatidyl serine Phosphatidyl inositol Phosphatidic acid Cardiolipin Sphingomyelin + phosphatidyl serine + phosphatidyl inositol Phosphatidic acid + Cardiolipin
‘A of lipid recovered from precipitate
3.6 4.1
0.9 1.4
6.1 8.5
1.9 2.8
8.2 7% 8.0
2.7 2.6 2%
8.4
2.5
8.3
2.8
23456769
5or
IO
II
II
I2
13
Fig. 3. Composition of rat tooth (enamel dentinc) total lipid and of bound lipid extracted from apatite precipitated by these lipids.
proportional to the amount of phospholipid added up to a maximal level of 150 mg per 100 ml of the solution (Fig. 1). Phospholipid caused the precipitatlon of approximately 40-50 per cent more apatite than the same weight of total lipid. Addition of total neutral lipids to metastable calcium phosphate solution produced no detectable precipitate. In the fourth experiment, individual commercially obtained lipids were added to metastable calcium phosphate solution; 150mg of lipid were added to IOOml of solution in each case. Only phospholipids produced a precipitate; the weights and percentage of lipid extracted after dissolving the precipitate in
23456769
IO
Lipids
12
13
acid are given in Table 3. Lecithin, sphingomyelin and phosphatidyl ethanolamine produced least but successively increasing weights of precipitate and less lecithin than sphingomyelin than phosphatidyl ethanolamine were incorporated into the precipitate. The remaining phospholipids produced very similar amounts of precipitate, approximately X.0 mg. containing similar amounts of bound lipid (2.7 per cent). Total lipids, total phospholipids and total neutral lipids were also used to promote precipitation from metastable solutions of calcium carbonate. Individual commercially obtained neutral and phospholipids were also used. In every case a precipitate was produced, weighing. 3450 mg/lOO ml of solution regardless of the amount or nature of the lipid added. No lipid could be detected in the precipitates after washing with chloroform-methanol. A number of the prccipitates were analysed by X-ray diffraction and shown to be calcite. The analysis of the various total lipids and the relative proportion of the lipids incorporated into the precipitated apatite are shown in Figs. 2~ 5. In the experiment using lipids from human and rat teeth some 6&70 per cent is neutral lipid, the remaining 3G40 per cent being phospholipid (Figs. 2 and 3). The composition of the lipid extracted from the precipitated apatite is quite different however, 1O- I5 per cent being neutral lipid and &90 per cent being phospholipid. The relative proportions of the phospholipids extracted from the precipitate are quite different from the proportions used to promote precipitation. Lecithin, calculated as a percentage of phospholipid, falls from 42 per cent in the human tooth lipid to 7.0 per cent in the lipid extracted frotn the
Lipids
Fig. 2. Composition of total lipid from human tooth (enamel dentine) and of bound lipid extracted from apatite precipitated from metastable calcium phosphate solution hy human tooth total lipids. Particular lipid classes arc numbered as follows in all the figures: (I) cholesterol; (2) cholesterol esters; (3) t’riglycerides; (4) monoglycerides; (5) diglycerides; (6) free fatty acids; (7) lecithin; (8) sphmgomyelin: (9) phosphatidyl ethanolamine; (10) phosphatidyl serine; (11) phosphatidyl inositol; (12) phosphatidie acid; (13) cardiolipin. The clear columns indicate analyses of the lipids used for precipitation; the dotted columns Indicate the analyses of the lipids extracted from the precipitated apatite after decalcification. The bar lines indicate the standard error of IO analyses in each case; where no bar line is drawn, the standard error of the mean was less than k 1 per cent.
5or
,I
123456 Lipids
Fig. 4. Composition of total lipid of rat liver and of bound lipid extracted from apatite precipitated by these lipids.
A. A. Odutuga, R. E. S.
314
Prout and R. .I. Hoare
Lipids
Fig. 5. Composition of total lipid bound lipid extracted from apatite lipids.
of rat kidney and of precipitated by these
decalcified precipitate, sphingomyelin from 10 to 7.0 per cent, phosphatidyl ethanolamine remains the same at 23 per cent and phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin increase from 5.0, 6.0, 6.0 and 7.0 per cent respectively to 15 per cent. The result with the lipids extracted from apatite precipitated by total lipids from rat teeth (Fig. 3) were similar. Similar differences between the proportions of the lipids added and those extracted from the precipitates were found when total lipids from rat liver and kidney were used (Figs. 4 and 5). The relative proportions of the lipids incorporated into apatite precipitated by phospholipids extracted from human teeth, rat teeth, liver and kidneys are shown in Figs. 69. The analysis of the phospholipids from each of the four tissues is also shown. The relative proportions of phospholipids incorporated into the precipitated apatite compared with the proportions of the phospholipids used to promote the precipitation are the same as those in the corresponding experiments using total lipids. In a further experiment, phospholipids containing [l-14C]-linoleic acid, which were obtained from liver and kidneys of rats previously injected with [ l-‘4C]linoleic acid, were used to promote precipitation of calcium phosphate from metastable solution. Eight experiments using 500 ml of metastable calcium phosphate solution were carried out; loo0 mg of phospholipid was used in four of the experiments and 800 mg in the remaining four. The precipitate was extracted, 60
Lipids
Fig. 7. Composition of rat tooth (enamelPdentine) total phospholipid and of bound lipid extracted from apatite precipitated by this lipid. Details as in Fig. 2, except that
seven analyses of the precipitates were carried out.
weighed and the lipid content measured as previously. The radioactivity of the lipid obtained after demineralization was determined and the specific activity in disintegrations per minute (dpm) per mg was obtained. The amount of precipitate and the percentage lipid was the same as in the previous experiments. The specific activity of the phospholipids was 98 dpm/ mg in both the lipid used to promote precipitation and that extracted from the precipitated apatite. DISCUSSION
Approximately 35 per cent of the lipid in enamel and dentine from both rat and human teeth is phospholipid (Prout et al., 1973; Odutuga and Prout, 1974). Lipids have been implicated in the mineralization process, particularly the acidic phosphoiipids (Takazoe et al., 1970; Irving, 1973a). In order to extract all the phospholipids from enamel and dentine it is necessary to demineralize the tissue with an acidic decalcifying agent before extraction with chloroform-methanol (2:1) (Shapiro et al., 1966; Shapiro. 1971; Prout et al., 1973). The phospholipids which are not soluble in this solvent until the calcified tissue has been demineralized with acid may be bound in some way to the inorganic phase or they may be bound to the enamel or dentine protein. Shapiro et al. (1966) working on bovine foetal dental tissues. 60.
I
50. 8
a-” 40
.-S .k ;:
30
E
20
40. ZJ ._ .c-
30-
;: $ u
s
IO
IO
0
zo-
7
8
9
IO
II
12
I3
Lipids
Fig. 6. Composition of human tooth (enamelLdentine) total phospholipid and of bound lipid extracted from apatite precipitated by this lipid.
oLipids
Fig. 8. Composition of total phospholipid of rat liver and of bound lipid extracted from apatite precipitated by this lipid.
Hydroxyapatite
precipitation
60
Lipids Fig. 9. Composition of total phospholipid of rat kidney and of bound lipid extracted from apatite precipitated by this lipid.
which are only slightly calcified, found that acid demineralization was necessary to release all the phospholipid. suggesting that some phospholipid may be protein-bound. The analysis of phospholipids from bovine foetal enamel treated with acid is very similar to the analysis obtained from rat mature enamel after demineralization (Prout et al., 1973) and rat enamel contains only approximately 1.0 per cent protein. Phospholipids extracted from Bacterionema mtruchotrr can precipitate hydroxyapatite from a metastable calcium phosphate solution in vitro (Takazoe uf (I/.. 1970). Joos and Carr (1967) and Cotmore et ul. (I Y7 1) have shown that phospholipids, particularly acidic phospholipids. will bind calcium in solution. Lipid analyses on rat and human enamel and dentine (Prout pt al.. 1973; Odutuga and Prout, 1974) have shown that acidic phospholipids, i.e. phosphatidyl serine. phosphatidyl inositol and phosphatidic acid. can only be extracted after demineralization of the tissues whereas lecithin, sphingomyelin and phosphatidyl ethanolamine are partially extractable from calcified tissues, the remainder only being extracted after decalcification. Cardiolipin, however, is completely extracted from calcified dentine but can only be extracted from decalcified enamel, which suggests a different binding in the two tissues. Liver and kidney total lipid contain much more phospholipid than total lipid from teeth, so it would be expected that the amount of apatite precipitated would be greater when total liver and kidney lipids are used than when teeth total lipids are used. However. the major components are lecithin and phosphatidy1 ethanolamine, which are not very effective in causing apatite precipitation, but a given weight of liver total lipid precipitates more apatite from 100 ml of metastable calcium phosphate solution than the same weight of total lipid from teeth (Fig. 1). The addition of total lipids, total neutral lipids or total phospholipids obtained from human teeth or rat teeth. liver or kidney to a metastable calcium carbonate solution precipitates calcite; the amount being constant from a given volume of solution, this is considered to be a seeding process. Individual commercial lipids produced the same effect. The precipitation of apatite was found to be directly proportional to the amount of phospholipid added to the metastable calcium phosphate solution
by lipids
315
up to a weight of &Omg of precipitate from 100 ml of metastable solution, the amount of phospholipid required to give a precipitate of 9.0 mg being 150 mg. When total lipid extract from the various tissues was used it was found that approximately 6 mg of precipitate was produced by 150mg of lipid, presumably owing to the presence of 6(t70 per cent neutral lipid which does not precipitate apatite. A molar ratio of calcium to phosphorus of 1.5-1.63 is considered to occur in apatite, depending on how it is formed (Irving, 1973b). In the present experiments the molar ratio of the calcium phosphate precipitate is 1.551.60 and this, in association with the X-ray analyses and the percentages of calcium and phosphate (Irving. 1973b), shows that the precipitate is apatite. The maximum amount of apatite precipitated from 100 ml of metastable calcium phosphate solution is 9.0 mg, which is less than the total amount of calcium phosphate present but presumably that remaining in solution is no longer metastable and further phospholipids will not cause precipitation. The total amount of calcium in 100 ml of metastable solution is 4QO8 mg and the weight of phosphorus 12.10 mg, the amount remaining after precipitation of 9.0mg of apatite will therefore be approximately 0.86 mg of calcium and IO.52 mg of phosphorus. From the above results it is therefore concluded that a specific reaction between phospholipid. calcium and phosphate is occurring which results in the formation of apatite. It has been suggested previously (Shapiro et ul., 1966; Prout et al.. 1973; Odutuga and Prout, 1974) that phospholipids, particularly acidic phospholipids, may be bound to the apatite of enamel and dentine as they can only be extracted after decalcification. The results in the present study support this idea as the extraction of apatite. precipitated from metastable calcium phosphate solution by phospholipids, using chloroform-methanol does not remove all the lipid. However. on dissolving the precipitated apatite. phospholipids are released and the major portion of the phospholipid is acidic phospholipid (Figs. 2-9). The fact that the relative proportions of the phospholipids bound to the precipitated apatite are quite different from the proportions used to promote precipitation is a further indication that a specific chemical reaction is occurring between the various phospholipids and calcium phosphate. If this were not so, it might be expected that the phospholipids would be present in the precipitated apatite in the same relative proportions as in the precipitating phospholipid mixture. The small amount of neutral lipid present in the apatite precipitated by the various total lipids (Figs. 2-5) is considered to be incorporated physically into the apatite crystals during their formation, as the amount is directly proportional to the amount of neutral lipid in the precipitating lipid and neutral lipids on their own did not precipitate apatite. When individual commercial phospholipids were used, lecithin, sphingomyelin and phosphatidyl ethanolamine were not only less effective in promoting precipitation but the amount of the individual phospholipid bound to the apatite as a percentage of the precipitate was also less than with the other phospholipids; lecithin being a less effective precipitating agent and being the least bound compared with
316
A. A. Odutuga, R. E. S. Prout and R. J. Hoare
sphingomyelin, phosphatidyl ethanolamine being the most effective of the three. Phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin precipitated very similar weights of apatite and formed a similar proportion of the apatite (Table 3). sphingomyelin, phosphatidyl ethanolLecithin, amine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid all have a molecular weight between 680 and 880 and contain one phosphate group, whereas cardiolipin has a molecular weight of about 1400 and contains two phosphate groups. A given weight of cardiolipin or other phospholipids will precipitate the same amount of apatite (Table 3); half the number of molecules of cardiolipin (containing two phosphates) being present compared with the number of molecules of the other phospholipids in a given weight. The effectiveness of the various phospholipids in promoting the precipitation of apatite from the metastable calcium phosphate solution would therefore appear to depend on the presence lecithin, of an acidic phosphate group, as sphingomyelin and phosphatidyl ethanolamine are the least effective in promoting precipitation. Studies of the lipids of matrix vesicles from bovine foetal epiphyseal cartilage carried out by Peress, Clarke Anderson and Sajdera (1974) show similar proportions of phospholipids associated with apatite to that found in the present study; they suggest that the vesicle phospholipids are acting as nucleation centres for apatite formation. It may be concluded from these experiments that phospholipids from either hard or soft tissues are able to precipitate apatite from a metastable calcium phosphate solution and that the acidic phospholipids are the most effective. The lipids in rat and human enamel and dentine are also mainly acidic phospholipids. The metastable solution has calcium and phosphate levels comparable to the ionic concentration found in saliva (Wasserman, Mandel and Levy, 1958) this may be quite different from that found at the calcifying sites in hard tissues. It is, however, suggested that in ~ivo phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin might be responsible for the initial precipitation of apatite during the formation of enamel and dentine, lecithin, sphingomyelin and phosphatidyl ethanolamine are also involved but may be less important. The neutral lipids in enamel and dentine would not appear to be involved in the initiation of calcihcation as they are not able to promote apatite precipitation in uitro.
Ackt~o~~/~rdyrrlzrr~ls~We wish to thank Mr. P. Hancock for skilled technical assistance, the Nigerian Government for provision of a Grant for A. A. Odutuga and Dr. S. L. Rowles of the Birmingham Dental School for confirmation of the X-ray crystallography results.
REFERENCES
Allred H. 1968. Investigations into the relationship between the lipid and other components of human dentine. Archs oral Biol. 13, 1077-1093. Bachra B. N., Trautz 0. R. and Simon S. L. 1963. Precipitation of calcium carbonates and phosphates-I: Spontaneous precipitation of calcium carbonates and phosphates under physiological conditions. Archs Biochem. 103, 124138. Cotmore J. M., Nichols G. and Wuthier R. E. 1971. Phospholipidcalcium phosphate complex: enhanced calcium migration in the presence of phosphate. Sciencr 172, 1339-1341. Fiske C. H. and Subbarow Y. 1925. The calorimetric determination of phosphorus. .I. biol. Chem. 66, 375-400. Folch J., Lees M. and Sloane-Stanley G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. hiol. Chem: 226, 4977509. Irving J. T. 1963. The sudanophil material at sites of calcification. Archs oral Biol. 8, 735745. Irving J. T. 1965. Lipids and calcifylaxis. Archs oral Biol. 10, 189-190. Irving J. T. 1973a. The pattern of sudanophilia in developing rat enamel. Archs oral Biol. 18. 137-140. Irving J. T. 1973b. Catciutn and Phosphorus Metnboiism, pp. 71-92. Academic Press, New York. Irving J. T. and Wuthier R. E. 1968. Histochemistry and biochemistry of calcification with special reference to the role of lipids. Clin. Orthop. 56, 237-260. Joos R. W. and Carr C. W. 1967. The binding of calcium in mixtures of phospholipids. Proc. Sot. rxp. Biol. Med. 124, 126% 1272. Kingsley G. R. and Robnet 0. 1957. New dye method for direct photometric determination of calcium. Am. 1. clin. Path. 27, 223-230. Odutuga A. A. and Prout R. E. S. 1973. Fatty acid composition of neutral lipids and phospholipids of enamel and dentine from rat incisors and molars. Archs oral Biol. 18, 689-697. Odutuga A. A. and Prout R. E. S. 1974. Lipid analysis of human enamel and dentine. Archs orul Biol. 19, 729731. Peress N. S., Clarke Anderson M. and Sajdera S. W. 1974. The lipids of matrix vesicles from bovine foetal epiphyseal cartilage. Cole. Tiss. Res. 14, 275-281. Prout R. E. S., Odutuga A. A. and Tring F. C. 1973. Lipid analysis of rat enamel and dentine. Archs oral Biol. 18, 373-380.
Prom R. E. S. and Odutuga A. A. 1974a. Fatty acid composition of neutral lipids and phospholipids of enamel and dentine from human molars. Archs orul Biol. 19, 293-298. Prout R. E. S. and Odutuga A. A. 1974b. In aiao incorporation of [1-“‘Cl-linoleic acid into the lipids of enamel and dentine of normal and essential fatty acid deficient rats. Archs oral Biol. 19, 1167-l170. Shapiro I. M. 1971. The neutral lipids of bovine bone. Archs oral Biol. 16, 41 I-421. Shapiro I. M., Wuthier R. E. and Irving J. T. 1966. A study of the phospholipids of bovine dental tissues-I: Enamel matrix and dentine. Archs oral Biol. 11, 501-5 12. Takazoe I., Vogel J. and Ennever J. 1970. Calcium hydroxyapatite nucleation by lipid extract of Bactrriowma matruchotii. J. dent. Res. 49, 395-398. Wasserman B. H., Mandel I. D. and Levy B. M. 195X. In vitro calcification of dental calculus. J. Periodont. 29, 144147.
Histochemical studies on the changes in the organic matrix of bone, dentine and enamel immediately before mineralization indicate that lipids (in particular phospholipids) may be involved in this process (Irving, 1963, 1965, 1973a; Irving and Wuthier, 1968). Histological staining of lipid in teeth and the extraction, particularly of phospholipid, occurs only after decalcification of the tissue (Allred, 1968; Shapiro, Wuthier and Irving, 1966; Prout, Odutuga and Tring, 1973). in uitro experiments have shown that phospholipids isolated from Bacterionma mtruchotii, a microorganism which forms apatite intracellularly, precipitate hydroxyapatite from a me&table solution of calcium phosphate (Takazoe, Vogel and Ennever, 1970). Cotmore, Nichols and Wuthier (1971) have shown that acidic phospholipids combine with calcium phosphate at pH 7.0 to form a precipitate of calcium phosphate and lipid from which the lipids could not be extracted by organic solvents. The purpose of the present study was to determine whether lipids extracted from hard and soft tissues could cause precipitation of hydroxyapatite from a metastable solution of calcium phosphate and, if so, to determine which particular lipids were responsible and the chemical nature of the precipitate. MATERIALS
AND
METHODS
Enamel-dentine mixtures were obtained from freshly extracted human molars and from the incisors and molars of 3-4-month-old Wistar rats; livers and kidneys were also obtained from the same rats which had been maintained on Oxoid modified diet 86 stock rat cubes (Herbert C. Styles (Bewdley) Ltd., Bewdley. Worcs.). The enamel-dentine samples were obtained and the lipids extracted after demineralization as deTable
I. Composition
Calcium phosphate solution in 0.04 M Verona1 buffer (PH 7.0) Calcium carbonate solution
of metastable
scribed by Prout rt al. (1973) and Prout and Odutuga (1974a). Liver and kidney lipids were extracted and purified by the method of Folch. Lees and SloaneStanley (1957). Neutral lipids and phospholipids from the various tissues were separated as described by Odutuga and Prout (1973). A metastable solution of calcium phosphate was prepared essentially by the method of Takazoe rt ~1. (1970) and of calcium carbonate as described by Bachra. Trautz and Simon (1963) (Table 1). To prevent bacterial contamination 200mg of thymol was added per litre of solution. Weighed quantities of either total lipid, neutral lipid, phospholipid or pure commercial lipids (Sigma Chemical Co., London, England) were added to the metastable solutions in conical flasks which were then sealed with Parafilm and aluminium foil. The mixtures were stirred, forming an emulsion, with magnetic stirrers for 7-21 days at room temperature. they were then centrifuged at 30,OOOg for 20 min at 5’C and any precipitate was collected. Precipitates were washed 4 times with distilled water and 3 times with 50ml of chloroform-~ methanol (2: 1, v/v) to remove lipid. The final residues were washed with ethyl alcohol, dried under nitrogen. weighed and stored at -20°C. Portions of the residues were dissolved in 5 N HCl and aliquots analysed for calcium (Kingsley and Robnet. 1957) and phosphorus (Fiske and Subbarow, 1925); the remaining acid solution was neutralized with NaHCO, and lipids were extracted and analysed qualitatively and quantitatively (Prout et al., 1973). Samples of the lipids extracted from the four different tissues (human enamel--dentine, rat enamel-dentine. liver and kidney) were also analysed (Prout cf al.. 1973; Prout and Odutuga, 1974a, b). X-ray diffraction analysis was carried out on the washed and dried precipitates. Samples were mounted
calcium phosphate
NaCl
KC1
70.0 145.0
4.7 5.0
and calcium carbonate
NaHCO,
22-o 22.0 III
Na2HP04
3-9 O-033
solutions
in mM
CaCI, .2Hz0
1a 75.0
pH
7.0
7.2 + 0.05
312
A. A. Odutuga, R. E. S. Prout and R. J. Hoare
Table 2. Composition
Source of lipid
of apatite
precipitated
No. of experiments
Total lipids Human teeth Rat teeth Rat liver Rat kidney Total phospholipids Human teeth Rat teeth Rat liver Rat kidney
from metastable calcium phosphate from different tissues
Lipid extracted from demineralized precipitate o/;,
solution
by lipids extracted
Ca/P in the precipitate (molar ratio)
Phosphorus in the precipitate
Calcium in the precipitate %
0,
/O
6 6 7 7
1.79 1.48 1.94 2.08
* * f +
0.21 0.28 0.26 026
35.61 35.97 34.96 35.66
+ f f +
064 0.35 0.39 0.24
17.97 f 17.41 i 17.72 + 17.65 f
0.09 0.18 033 0.22
1.54 1.60 1.53 1.57
* F * *
0.04 0.02 0.04 003
3 4 3 3
I.17 1.68 I.86 1.67
+ f + k
0.20 0.14 052 0.29
34.91 36.21 35.39 35.54
k + f +
0.61 0.21 0.45 0.45
17.48 17.27 17.25 17.82
0.22 0.16 0.15 0.19
1.56 1.63 1.59 I.55
+ + + +
0.02 0.02 0.06 0.02
in 0.5 mm quartz capillaries and X-ray patterns were recorded using filtered copper radiation and a flat plate camera. Phospholipids containing [l-‘4C]-linoleic acid were obtained from livers and kidneys of 17 normal 3-4-month-old Wistar rats that had been injected 8 days previously with 750 &i of [l-‘“Cl-linoleic acid. The radioactivity of the phospholipid was determined in disintegrations per minute (dpm) per mg as previously described (Prout and Odutuga, 1974b). Radioactive phospholipids were added to metastable calcium phosphate solution as described above. RESULTS
The total lipids extracted from human teeth, rat teeth, liver and kidneys caused the precipitation of calcium phosphate from the metastable solution. The percentages of calcium and phosphate in the precipitates are shown in Table 2, together with the calciumphosphorus molar ratio. X-ray analysis of these precipitates shows them to be apatite, except in one sample precipitated by rat liver total lipid and one sample precipitated by rat kidney total lipid. The weight of apatite precipitated from the metastable calcium phosphate solution is directly proportional to the amount of total lipid added, up to approximately 150mg lipid/lOOml of solution (Fig. 1). The weight of precipitate is slightly lower when the total lipids extracted from human and rat teeth were used than with total lipids from rat liver and kidney. X0 mg/lOO ml very little further precipitation
In the second experiment, total lipids were extracted from human teeth and rat teeth, liver and kidneys and separated into two fractions, one containing total neutral lipid and free fatty acids and the other total phospholipid. The total phospholipids were added to the metastable calcium phosphate solution, and precipitates formed with the composition shown in Table 2 and which X-ray analysis showed to be apatite. The weight of precipitate was slightly higher with total phospholipids from human and rat teeth than when phospholipids from rat liver and kidney were used to promote precipitation (Fig. 1). The amount of phospholipid incorporated into the precipitated apatite varied between 1.2-1.9 per cent (Table 2), depending on the origin of the phospholipid. As with total lipid, the weight of precipitate was directly
Above
occurs. The apatite crystals precipitated by the total lipids from the four tissues contain lipid, which could not be removed by washing with chloroform-methanol unless the precipitate was dissolved in acid when lipid was released which could be extracted from the solution. The lipid bound to the precipitated apatite is shown in Table 2 as a percentage by weight of the precipitate, the percentage being between 1.5-2.0, depending on the origin of the lipid used to promote precipitation.
k + k f
I 0
-
1
,
50
100
I
I50
1
200
mg lipid Fig. 1. Weight of apatite (including bound lipid) precipitated from 100 ml of metastable calcium phosphate solution by differing amounts of lipid from different sources as follows: A, total phospholipid extracted from rat teeth; a, total phospholipid extracted from human teeth; 0, total phospholipid extracted from rat kidney; n, total phospholipid extracted from rat liver; 0, total lipid extracted from rat liver; 0, total lipid extracted from rat kidney; @, total lipid extracted from rat teeth;m, total lipid extracted from human teeth.
Hydroxyapatite
precipitation
by lipids
Table 3. Precipitation of apatite from 100 ml of metastable calcium phosphate solution using 150 mg of phospholipid in each case.
wt. of precipitate (mg) Lecithin Sphingomyelin Phosphatidyl ethanolamine Phosphatidyl serine Phosphatidyl inositol Phosphatidic acid Cardiolipin Sphingomyelin + phosphatidyl serine + phosphatidyl inositol Phosphatidic acid + Cardiolipin
‘A of lipid recovered from precipitate
3.6 4.1
0.9 1.4
6.1 8.5
1.9 2.8
8.2 7% 8.0
2.7 2.6 2%
8.4
2.5
8.3
2.8
23456769
5or
IO
II
II
I2
13
Fig. 3. Composition of rat tooth (enamel dentinc) total lipid and of bound lipid extracted from apatite precipitated by these lipids.
proportional to the amount of phospholipid added up to a maximal level of 150 mg per 100 ml of the solution (Fig. 1). Phospholipid caused the precipitatlon of approximately 40-50 per cent more apatite than the same weight of total lipid. Addition of total neutral lipids to metastable calcium phosphate solution produced no detectable precipitate. In the fourth experiment, individual commercially obtained lipids were added to metastable calcium phosphate solution; 150mg of lipid were added to IOOml of solution in each case. Only phospholipids produced a precipitate; the weights and percentage of lipid extracted after dissolving the precipitate in
23456769
IO
Lipids
12
13
acid are given in Table 3. Lecithin, sphingomyelin and phosphatidyl ethanolamine produced least but successively increasing weights of precipitate and less lecithin than sphingomyelin than phosphatidyl ethanolamine were incorporated into the precipitate. The remaining phospholipids produced very similar amounts of precipitate, approximately X.0 mg. containing similar amounts of bound lipid (2.7 per cent). Total lipids, total phospholipids and total neutral lipids were also used to promote precipitation from metastable solutions of calcium carbonate. Individual commercially obtained neutral and phospholipids were also used. In every case a precipitate was produced, weighing. 3450 mg/lOO ml of solution regardless of the amount or nature of the lipid added. No lipid could be detected in the precipitates after washing with chloroform-methanol. A number of the prccipitates were analysed by X-ray diffraction and shown to be calcite. The analysis of the various total lipids and the relative proportion of the lipids incorporated into the precipitated apatite are shown in Figs. 2~ 5. In the experiment using lipids from human and rat teeth some 6&70 per cent is neutral lipid, the remaining 3G40 per cent being phospholipid (Figs. 2 and 3). The composition of the lipid extracted from the precipitated apatite is quite different however, 1O- I5 per cent being neutral lipid and &90 per cent being phospholipid. The relative proportions of the phospholipids extracted from the precipitate are quite different from the proportions used to promote precipitation. Lecithin, calculated as a percentage of phospholipid, falls from 42 per cent in the human tooth lipid to 7.0 per cent in the lipid extracted frotn the
Lipids
Fig. 2. Composition of total lipid from human tooth (enamel dentine) and of bound lipid extracted from apatite precipitated from metastable calcium phosphate solution hy human tooth total lipids. Particular lipid classes arc numbered as follows in all the figures: (I) cholesterol; (2) cholesterol esters; (3) t’riglycerides; (4) monoglycerides; (5) diglycerides; (6) free fatty acids; (7) lecithin; (8) sphmgomyelin: (9) phosphatidyl ethanolamine; (10) phosphatidyl serine; (11) phosphatidyl inositol; (12) phosphatidie acid; (13) cardiolipin. The clear columns indicate analyses of the lipids used for precipitation; the dotted columns Indicate the analyses of the lipids extracted from the precipitated apatite after decalcification. The bar lines indicate the standard error of IO analyses in each case; where no bar line is drawn, the standard error of the mean was less than k 1 per cent.
5or
,I
123456 Lipids
Fig. 4. Composition of total lipid of rat liver and of bound lipid extracted from apatite precipitated by these lipids.
A. A. Odutuga, R. E. S.
314
Prout and R. .I. Hoare
Lipids
Fig. 5. Composition of total lipid bound lipid extracted from apatite lipids.
of rat kidney and of precipitated by these
decalcified precipitate, sphingomyelin from 10 to 7.0 per cent, phosphatidyl ethanolamine remains the same at 23 per cent and phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin increase from 5.0, 6.0, 6.0 and 7.0 per cent respectively to 15 per cent. The result with the lipids extracted from apatite precipitated by total lipids from rat teeth (Fig. 3) were similar. Similar differences between the proportions of the lipids added and those extracted from the precipitates were found when total lipids from rat liver and kidney were used (Figs. 4 and 5). The relative proportions of the lipids incorporated into apatite precipitated by phospholipids extracted from human teeth, rat teeth, liver and kidneys are shown in Figs. 69. The analysis of the phospholipids from each of the four tissues is also shown. The relative proportions of phospholipids incorporated into the precipitated apatite compared with the proportions of the phospholipids used to promote the precipitation are the same as those in the corresponding experiments using total lipids. In a further experiment, phospholipids containing [l-14C]-linoleic acid, which were obtained from liver and kidneys of rats previously injected with [ l-‘4C]linoleic acid, were used to promote precipitation of calcium phosphate from metastable solution. Eight experiments using 500 ml of metastable calcium phosphate solution were carried out; loo0 mg of phospholipid was used in four of the experiments and 800 mg in the remaining four. The precipitate was extracted, 60
Lipids
Fig. 7. Composition of rat tooth (enamelPdentine) total phospholipid and of bound lipid extracted from apatite precipitated by this lipid. Details as in Fig. 2, except that
seven analyses of the precipitates were carried out.
weighed and the lipid content measured as previously. The radioactivity of the lipid obtained after demineralization was determined and the specific activity in disintegrations per minute (dpm) per mg was obtained. The amount of precipitate and the percentage lipid was the same as in the previous experiments. The specific activity of the phospholipids was 98 dpm/ mg in both the lipid used to promote precipitation and that extracted from the precipitated apatite. DISCUSSION
Approximately 35 per cent of the lipid in enamel and dentine from both rat and human teeth is phospholipid (Prout et al., 1973; Odutuga and Prout, 1974). Lipids have been implicated in the mineralization process, particularly the acidic phosphoiipids (Takazoe et al., 1970; Irving, 1973a). In order to extract all the phospholipids from enamel and dentine it is necessary to demineralize the tissue with an acidic decalcifying agent before extraction with chloroform-methanol (2:1) (Shapiro et al., 1966; Shapiro. 1971; Prout et al., 1973). The phospholipids which are not soluble in this solvent until the calcified tissue has been demineralized with acid may be bound in some way to the inorganic phase or they may be bound to the enamel or dentine protein. Shapiro et al. (1966) working on bovine foetal dental tissues. 60.
I
50. 8
a-” 40
.-S .k ;:
30
E
20
40. ZJ ._ .c-
30-
;: $ u
s
IO
IO
0
zo-
7
8
9
IO
II
12
I3
Lipids
Fig. 6. Composition of human tooth (enamelLdentine) total phospholipid and of bound lipid extracted from apatite precipitated by this lipid.
oLipids
Fig. 8. Composition of total phospholipid of rat liver and of bound lipid extracted from apatite precipitated by this lipid.
Hydroxyapatite
precipitation
60
Lipids Fig. 9. Composition of total phospholipid of rat kidney and of bound lipid extracted from apatite precipitated by this lipid.
which are only slightly calcified, found that acid demineralization was necessary to release all the phospholipid. suggesting that some phospholipid may be protein-bound. The analysis of phospholipids from bovine foetal enamel treated with acid is very similar to the analysis obtained from rat mature enamel after demineralization (Prout et al., 1973) and rat enamel contains only approximately 1.0 per cent protein. Phospholipids extracted from Bacterionema mtruchotrr can precipitate hydroxyapatite from a metastable calcium phosphate solution in vitro (Takazoe uf (I/.. 1970). Joos and Carr (1967) and Cotmore et ul. (I Y7 1) have shown that phospholipids, particularly acidic phospholipids. will bind calcium in solution. Lipid analyses on rat and human enamel and dentine (Prout pt al.. 1973; Odutuga and Prout, 1974) have shown that acidic phospholipids, i.e. phosphatidyl serine. phosphatidyl inositol and phosphatidic acid. can only be extracted after demineralization of the tissues whereas lecithin, sphingomyelin and phosphatidyl ethanolamine are partially extractable from calcified tissues, the remainder only being extracted after decalcification. Cardiolipin, however, is completely extracted from calcified dentine but can only be extracted from decalcified enamel, which suggests a different binding in the two tissues. Liver and kidney total lipid contain much more phospholipid than total lipid from teeth, so it would be expected that the amount of apatite precipitated would be greater when total liver and kidney lipids are used than when teeth total lipids are used. However. the major components are lecithin and phosphatidy1 ethanolamine, which are not very effective in causing apatite precipitation, but a given weight of liver total lipid precipitates more apatite from 100 ml of metastable calcium phosphate solution than the same weight of total lipid from teeth (Fig. 1). The addition of total lipids, total neutral lipids or total phospholipids obtained from human teeth or rat teeth. liver or kidney to a metastable calcium carbonate solution precipitates calcite; the amount being constant from a given volume of solution, this is considered to be a seeding process. Individual commercial lipids produced the same effect. The precipitation of apatite was found to be directly proportional to the amount of phospholipid added to the metastable calcium phosphate solution
by lipids
315
up to a weight of &Omg of precipitate from 100 ml of metastable solution, the amount of phospholipid required to give a precipitate of 9.0 mg being 150 mg. When total lipid extract from the various tissues was used it was found that approximately 6 mg of precipitate was produced by 150mg of lipid, presumably owing to the presence of 6(t70 per cent neutral lipid which does not precipitate apatite. A molar ratio of calcium to phosphorus of 1.5-1.63 is considered to occur in apatite, depending on how it is formed (Irving, 1973b). In the present experiments the molar ratio of the calcium phosphate precipitate is 1.551.60 and this, in association with the X-ray analyses and the percentages of calcium and phosphate (Irving. 1973b), shows that the precipitate is apatite. The maximum amount of apatite precipitated from 100 ml of metastable calcium phosphate solution is 9.0 mg, which is less than the total amount of calcium phosphate present but presumably that remaining in solution is no longer metastable and further phospholipids will not cause precipitation. The total amount of calcium in 100 ml of metastable solution is 4QO8 mg and the weight of phosphorus 12.10 mg, the amount remaining after precipitation of 9.0mg of apatite will therefore be approximately 0.86 mg of calcium and IO.52 mg of phosphorus. From the above results it is therefore concluded that a specific reaction between phospholipid. calcium and phosphate is occurring which results in the formation of apatite. It has been suggested previously (Shapiro et ul., 1966; Prout et al.. 1973; Odutuga and Prout, 1974) that phospholipids, particularly acidic phospholipids, may be bound to the apatite of enamel and dentine as they can only be extracted after decalcification. The results in the present study support this idea as the extraction of apatite. precipitated from metastable calcium phosphate solution by phospholipids, using chloroform-methanol does not remove all the lipid. However. on dissolving the precipitated apatite. phospholipids are released and the major portion of the phospholipid is acidic phospholipid (Figs. 2-9). The fact that the relative proportions of the phospholipids bound to the precipitated apatite are quite different from the proportions used to promote precipitation is a further indication that a specific chemical reaction is occurring between the various phospholipids and calcium phosphate. If this were not so, it might be expected that the phospholipids would be present in the precipitated apatite in the same relative proportions as in the precipitating phospholipid mixture. The small amount of neutral lipid present in the apatite precipitated by the various total lipids (Figs. 2-5) is considered to be incorporated physically into the apatite crystals during their formation, as the amount is directly proportional to the amount of neutral lipid in the precipitating lipid and neutral lipids on their own did not precipitate apatite. When individual commercial phospholipids were used, lecithin, sphingomyelin and phosphatidyl ethanolamine were not only less effective in promoting precipitation but the amount of the individual phospholipid bound to the apatite as a percentage of the precipitate was also less than with the other phospholipids; lecithin being a less effective precipitating agent and being the least bound compared with
316
A. A. Odutuga, R. E. S. Prout and R. J. Hoare
sphingomyelin, phosphatidyl ethanolamine being the most effective of the three. Phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin precipitated very similar weights of apatite and formed a similar proportion of the apatite (Table 3). sphingomyelin, phosphatidyl ethanolLecithin, amine, phosphatidyl serine, phosphatidyl inositol, phosphatidic acid all have a molecular weight between 680 and 880 and contain one phosphate group, whereas cardiolipin has a molecular weight of about 1400 and contains two phosphate groups. A given weight of cardiolipin or other phospholipids will precipitate the same amount of apatite (Table 3); half the number of molecules of cardiolipin (containing two phosphates) being present compared with the number of molecules of the other phospholipids in a given weight. The effectiveness of the various phospholipids in promoting the precipitation of apatite from the metastable calcium phosphate solution would therefore appear to depend on the presence lecithin, of an acidic phosphate group, as sphingomyelin and phosphatidyl ethanolamine are the least effective in promoting precipitation. Studies of the lipids of matrix vesicles from bovine foetal epiphyseal cartilage carried out by Peress, Clarke Anderson and Sajdera (1974) show similar proportions of phospholipids associated with apatite to that found in the present study; they suggest that the vesicle phospholipids are acting as nucleation centres for apatite formation. It may be concluded from these experiments that phospholipids from either hard or soft tissues are able to precipitate apatite from a metastable calcium phosphate solution and that the acidic phospholipids are the most effective. The lipids in rat and human enamel and dentine are also mainly acidic phospholipids. The metastable solution has calcium and phosphate levels comparable to the ionic concentration found in saliva (Wasserman, Mandel and Levy, 1958) this may be quite different from that found at the calcifying sites in hard tissues. It is, however, suggested that in ~ivo phosphatidyl serine, phosphatidyl inositol, phosphatidic acid and cardiolipin might be responsible for the initial precipitation of apatite during the formation of enamel and dentine, lecithin, sphingomyelin and phosphatidyl ethanolamine are also involved but may be less important. The neutral lipids in enamel and dentine would not appear to be involved in the initiation of calcihcation as they are not able to promote apatite precipitation in uitro.
Ackt~o~~/~rdyrrlzrr~ls~We wish to thank Mr. P. Hancock for skilled technical assistance, the Nigerian Government for provision of a Grant for A. A. Odutuga and Dr. S. L. Rowles of the Birmingham Dental School for confirmation of the X-ray crystallography results.
REFERENCES
Allred H. 1968. Investigations into the relationship between the lipid and other components of human dentine. Archs oral Biol. 13, 1077-1093. Bachra B. N., Trautz 0. R. and Simon S. L. 1963. Precipitation of calcium carbonates and phosphates-I: Spontaneous precipitation of calcium carbonates and phosphates under physiological conditions. Archs Biochem. 103, 124138. Cotmore J. M., Nichols G. and Wuthier R. E. 1971. Phospholipidcalcium phosphate complex: enhanced calcium migration in the presence of phosphate. Sciencr 172, 1339-1341. Fiske C. H. and Subbarow Y. 1925. The calorimetric determination of phosphorus. .I. biol. Chem. 66, 375-400. Folch J., Lees M. and Sloane-Stanley G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissues. J. hiol. Chem: 226, 4977509. Irving J. T. 1963. The sudanophil material at sites of calcification. Archs oral Biol. 8, 735745. Irving J. T. 1965. Lipids and calcifylaxis. Archs oral Biol. 10, 189-190. Irving J. T. 1973a. The pattern of sudanophilia in developing rat enamel. Archs oral Biol. 18. 137-140. Irving J. T. 1973b. Catciutn and Phosphorus Metnboiism, pp. 71-92. Academic Press, New York. Irving J. T. and Wuthier R. E. 1968. Histochemistry and biochemistry of calcification with special reference to the role of lipids. Clin. Orthop. 56, 237-260. Joos R. W. and Carr C. W. 1967. The binding of calcium in mixtures of phospholipids. Proc. Sot. rxp. Biol. Med. 124, 126% 1272. Kingsley G. R. and Robnet 0. 1957. New dye method for direct photometric determination of calcium. Am. 1. clin. Path. 27, 223-230. Odutuga A. A. and Prout R. E. S. 1973. Fatty acid composition of neutral lipids and phospholipids of enamel and dentine from rat incisors and molars. Archs oral Biol. 18, 689-697. Odutuga A. A. and Prout R. E. S. 1974. Lipid analysis of human enamel and dentine. Archs orul Biol. 19, 729731. Peress N. S., Clarke Anderson M. and Sajdera S. W. 1974. The lipids of matrix vesicles from bovine foetal epiphyseal cartilage. Cole. Tiss. Res. 14, 275-281. Prout R. E. S., Odutuga A. A. and Tring F. C. 1973. Lipid analysis of rat enamel and dentine. Archs oral Biol. 18, 373-380.
Prom R. E. S. and Odutuga A. A. 1974a. Fatty acid composition of neutral lipids and phospholipids of enamel and dentine from human molars. Archs orul Biol. 19, 293-298. Prout R. E. S. and Odutuga A. A. 1974b. In aiao incorporation of [1-“‘Cl-linoleic acid into the lipids of enamel and dentine of normal and essential fatty acid deficient rats. Archs oral Biol. 19, 1167-l170. Shapiro I. M. 1971. The neutral lipids of bovine bone. Archs oral Biol. 16, 41 I-421. Shapiro I. M., Wuthier R. E. and Irving J. T. 1966. A study of the phospholipids of bovine dental tissues-I: Enamel matrix and dentine. Archs oral Biol. 11, 501-5 12. Takazoe I., Vogel J. and Ennever J. 1970. Calcium hydroxyapatite nucleation by lipid extract of Bactrriowma matruchotii. J. dent. Res. 49, 395-398. Wasserman B. H., Mandel I. D. and Levy B. M. 195X. In vitro calcification of dental calculus. J. Periodont. 29, 144147.
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