A study of the phospholipids of bovine dental tissues—I

Arch. oral Bid.

Vol.1 I, pp.SOl-512, 1966. Pergamon Press Ltd.Printed inGt.Britain.

A STUDY OF THE PHOSPHOLIPIDS DENTAL TISSUES-I ENAMEL

OF BOVINE

MATRIX AND DENTINE

I. M. SHAPIRO, R. E. WUTHIER and J. T. IRVING Forsyth Dental Center, 140 The Fenway, Boston, Mass. 02115, U.S.A. Summary-The phospholipids of bovine foetal enamel matrix and dentine extracted before and after demineralization were examined. Enamel matrix contained a much higher percentage of lipid than dentine. Before deminerahzation only 40 per cent of the total phospholipid could be extracted from enamel matrix, whereas over 2/3 of the total could be extracted from dentine. In both tissues, neutral phosphatides were extracted primarily before demineralization, whilst the acidic phosphatides were At each extraction stage the phosphatides extracted only after demineralization. liberated from both enamel matrix and dentine were similar qualitatively, but differed in amounts and proportions. It is suggested that the acidic phosphatides may play a role in the calcification process. INTRODUCTION

A REVIEWof the literature of the last 30 years revealed a paucity of information regarding the lipids of the dental tissues. AKAMATSU (1929) drew attention to the fact that lipids were present in the calcified dental tissues. WISLOCKI, SINGER and WALDO (1948) and WISLOCKI and SOGNNAES(1950) demonstrated lipoidic material in the prism sheaths, the interprismatic substance and cross striations of enamel. KRASNOW (1934) using the phosphorus content of lipids as a quantitative guide, estimated that enamel and dentine contained 2-8 and l-7 per cent of lipid respectively. HESS,LEEand PECKHAM(1956) found that dentine contained 0.014 per cent and enamel 0,075 per cent phospholipid. Measurements of the cholesterol content of dentine was made by LEOPOLD, HESS and CARTER(1951). SOYENKOFF, FRIEDMANand NEWTON (1951) described the occurrence of an amino phospholipid in whole tooth. The presence of choline containing phospholipids in the peritubular dentine was reported by ALLRED(1964), whilst STEWART,CLAIBOURNE and LUIKART(1965) described a high concentration of phospholipids in the odontoblastic layer of human dentine. DIRKSEN(1963) using newly developed techniques of lipid separation, showed that the phospholipid components of dentine consisted of phosphatidylinositol, sphingomyelin, lecithin, phosphatidylethanolamine and lyso cephalin (lyso phosphatidylethanolamine ?). He also detected three unidentified lipids which he tentatively proposed to be polyglycerol phosphatides (cardiolipin) and, or, phosphatidic acids. DIRKSENand IKELS(1964) using improved techniques carried out a quantitative study of the total lipid content of human dentine and showed that phospholipids accounted for 18 per cent of the total lipids present. He also measured the phospholipid content of dentine after decalcification with EDTA (ethylenediaminetetraacetic acid). There 501

502

I. M. SHAPIRO, R.E. WUTHIER AND

.I.T.IRVING

no attempt, however, to quantitativly estimate the individual phospholipids present in the polar lipid fraction. SYMONS (1958) and SUGA(1959) described a heavy concentration of phospholipid in the ameloblastic layer of the enamel matrix. The authors have found no evidence in the literature of any analyses per se carried out on the component phosphatides of the enamel matrix. IRVING(1958) reported a method of staining hard tissues in which calcification was being initiated or proceeding. He found that after extracting the tissue with lipid solvents and then staining the sections with Sudan Black the calcifying matrix showed intense sudanophilia. WUTHIER and IRVING (1964) presented evidence that the sudanophilic substance was a lipid which was acidic in nature. JOHNSON(1960) reported that the phosphatides disappeared just before calcification occurs. Cmuss and CLARK(1965) maintained rats on diets containing an excess of vitamin D and found that there was a raised phospholipid level in the bones; they too suggested that the phosphatides are concerned in ossification. THOMPSONand DELUCA (1964) demonstrated a relationship between vitamin D and phospholipid metabolism. HOSOYA,WATANABEand FUJIMORI(1964), working in vitro and in viva showed that vitamin D, is an essential co-factor in the incorporation of serine into phosphatidylserine. In view of the incrimination of the phospholipids with the calcification mechanism, a study of the nature and quantity of the phosphatides present in the enamel matrix (a calcifying tissue) and dentine (a calcified tissue) was carried out. was

EXPERIMENTAL METHODS Bovine embryos aged about 6 months in utero were obtained from an abbatoir. The foetuses were removed from their maternal membranes soon after death and exsanguination of the parent, the lower jaw detached from the foetus by guillotining through the mandible posterior to the developing canine tooth. This was then removed to an insulated container and stored frozen with solid CO,. The jaws were thawed in a saline solution (0.9% NaCl) and maintained at 0°C throughout the dissection. Sudan Black staining of foetal bovine jaws

Bovine foetal jaws after 3, 6 and 9 months development in utero were examined histochemically to determine whether the sudanophilic reaction described by IRVING (1963) occurred in the enamel matrix of the incisor teeth of the calf. The jaws were fixed in 5 % formal-saline for at least 2 days and processed histochemically according to IRVING(1963). Stained sections were subjected to a further extraction using 0.3 % HCl in absolute alcohol for 16 hr. They were restained to determine if this latter procedure removed the sudanophilic material. Preparation of the tissues

The mucosa overlying the incisor teeth was incised, the teeth enucleated and the dental pulps removed. The teeth were cleaned of adherent soft tissue, the surface of the teeth being scraped gently with a small ignition file to remove the outer cementum

ENAMELMATRIX

ANDDENTINEPHosPHATILIW:

503

layer (L EVINE,GLIMCHERand BONAR,1964) and then washed in a stream of freezing saline. The cheese-like enamel matrix was removed from the teeth with a blunt dental chisel, care was taken not to remove matrix in the region of the dento-enamel junction. The dentine was cut from the developing root, prior to being vigourously filed in order to remove the cementum. The enamel matrix and the dentine were washed thrice with saline and water (at OOC).The dentine was frozen in solid CO, and milled to a powder which would pass through a 60 mesh sieve. The enamel matrix and dentine were lyophilized and then weighed. First extraction

The freeze dried enamel matrix and dentine were extracted with 250 ml of chloroform-methanol, 2/l, v/v. (C/M) for 3 days at room temperature in an atmosphere of nitrogen. Extraction was aided by agitation on a Wrist Action Shaker. The tissues were then homogenized with C/M in a ground glass homogenizer to ensure penetration of the lipid solvents into the tissues. The tissues were separated from the solvent extract by centrifugation, dried and weighed. Second rxtruction

The enamel matrix and dentine were demineralized with 0.5 M EDTA at pH X.2 for 3 days and then dialysed against running tap water for 72 hr. The extract was concentrated by rotary evaporation, lyophilized and weighed. The tissues were then extracted in a manner similar to the first extraction. Third extruction

The residue of the second extraction was dried under vacuum, weighed and then re-extracted with acidified solvent (C/M/cone HCI ; 200/100/l, v/v); the residue was discarded. The extract was brought to neutral pH by the addition of finely powdered NaHCO, and the sediment was removed by filtration. Detection

of u mrthunol solulde enumel protein

A large quantity of insoluble residue was extracted at the second extraction stage. This was collected by filtration and found to be partly soluble in methanol. The methanol soluble extract scanned on a Beckman DU Recording spectrophotometer from 200 to 600 rnp. Purijicution und separution of’ the lipid extructs Sephadex chromatography. This was the method reported

by WUTHIER(1966a) and was used to separate lipid from non-lipid contaminants. A saturated mixture of C/M/water(200/100/75, v/v) was prepared and the two phases allowed to separate. Sephadex G25 (beads) was allowed to swell overnight in the upper methanol/water phase and then poured into a 1x 10 cm chromatography column. The dried crude lipid extract was dissolved in a small volume of the lower C/M phase, applied to the column. The column was then eluted with 25 ml of the lower phase under N, pressure.

I. M. SHAPIRO, K. E. WUTHIER AND J. T. IRVING

so-4

The effluant was concentrated by evaporation in a stream of N, and taken to complete dryness under high vacuum. The dried lipid was then dissolved in a small volume of chloroform for silicic acid chromatography. Silicic ucid chromutogruphy. This was used to separate the lipid extracts into polar and non-polar fractions. A thick slurry of silicic acid (Mallinckdrodt, 100 mesh) in chloroform was packed (under N2) into a chromatography column (1 x 10 cm). The silicic acid was washed successively with 25-ml portions of chloroform, C/M and chloroform. The lipid was applied to the column in chloroform, the nonpolar lipids being eluted from the column with 25 ml of chloroform, the polar lipids with 50 ml of methanol. The effluants were evaporated to dryness and weighed. Phospholipid paper chroma rography . This was the method described by WUTHIER (1966b). The polar lipids were dissolved in C/M, 10 pg of lipid/$ of solvent and 200-300 pg of lipid was applied to the chromatography paper. Chromatography was carried out in two solvent directions. C’hronwtography paper. Whatman S.G. 81 (Available from H. Reeve Angel Inc., Clifton, New Jersey). This was cut into rectangles 6 I. 7.5 in.: the larger side being used in the direction of the first solvent system. Chromatography chambers. Three-litre, brown glass bottles, 10 in. high, 5 in. diameter, with a 3-in. diameter neck. Solvent systems 1Di-isobutyl ketone I Glacial acetic acid

First dimension

Chloroform Methanol Water

45 ml 25 ml 23 ml 10 ml 4 ml

Pyridine 34 ml Chloroform 32 ml Di-isobutyl ketone 25 ml Second dimension 17.5 ml Methanol i 0.5 M NH,Cl pH 10.4 6 ml All solvents were redistilled before use. Appkcation of lipid to chromatography puper. A known quantity of lipid dissolved in C/M was applied to the paper and dried in a stream of N,. The papers were rolled into cylinders, developed lengthwise in the first solvent system. dried for 30 min in air and rotated through 90” for the second system. The solvents were allowed to reach a few mm from the top of the paper in both systems, before being removed from the bottles and allowed to dry. The rolled papers maintained their shape through stainless steel Clips.

ENAMEL MATRIX AND DENTINE PHOSPHATJDES

505

Staining of the chromatograms. Each paper was washed with tapwater, O*lM acetic acid and re-washed with deionized water. The chromatograms were stained with Rhodamine 6G (MARINETTI,1962) for 5-10 min and re-washed with water. The papers were viewed in U.V.light at 360 rnp whilst still wet and observed periodically until dry, the areas occupied by the different phospholipids being outlined in pencil. The papers were then sprayed with ninhydrin (MARINIXTI, 1962). Phosphorus analysis. Each spot was cut from the paper, weighed, digested with 70 % perchloric acid and then analysed for phosphorus by a modification of the method of MARTINand DOTY(1949). A correction was made for the background phosphorus of the paper.

RESULTS Weight of extracted phospholipid

Application of the conversion factor P x 25 permitted the weight of lipid extracted from the tissues to be expressed as milligrams of phosphatide per 100 g of dried tissue. In Table 1, it can be seen that the total weight of phospholipid extracted from both enamel matrix and dentine was low compared with other tissues. Expressed on the basis of the original dry weight, enamel matrix contained about 20 times more lipid than dentine; expressed on the basis of the demineralized weight, enamel matrix contained 3-4 times more lipid. After demineralization 10 times more lipid was extracted from enamel matrix than from dentine matrix. Little additional lipid could be removed from either matrix when further extracted with acidified lipid solvent. Phosphorus analysis of chromatograms of the enamel matrix

In Table 2, the results of the analysis of the individual phospholipids can be seen expressed as a percentage of the total pg of lipid P in each chromatogram. Extracts of undemineralized enamel reveal three major components identified as sphingomyelin (Sph), lecithin (Let) and phosphatidylethanolamine (PE). Lecithin (Let) is the major fraction; Sph and PE occupy the remaining 25 per cent. The mean total lipid phosphorus at this stage was 95 pg/g of dry undemineralized tissue. Demineralized enamel matrix contained four major components; PE, phosphatidylinositol (PI), phosphatidylserine (PS), and cardiolipin (CL). About half of the lipid phosphorus was found in PE; the acidic phosphatides PI, PS and CL (not extractable until this stage) represented the bulk of the remaining phospholipids. Phosphatidic acid (PA), lyso PE and Let were present in lesser and more variable amounts. The mean total lipid phosphorus extracted after demineralization was 106.1 pg/g of dry demineralized enamel. Chromatograms of acid extracted enamel matrix showed a somewhat similar pattern of phosphatides as the demineralized extracts. They were in much smaller quantities and PE, PA and CL were no longer seen. Lecithin was found to be the largest fraction, followed by Sph, PS and PI (in that order). The mean total lipid phosphorus was 7.8 pg/g of dry demineralized lipid extracted enamel matrix.

3100

3400

(A)

(B)

2390

Dentine

3100

(A)

Tissue

Tissue weight (mg)

19.4

21.3

725

600

28.2

21.6

648

670

Phospholipid TX: (mg/lOOg) m

Before demineralizatton

925

794

244

399

Tissue weight (mg)

275

227

700

1020

29.8

28.5

286.7

256.0

Phospholipid

After demineralization

917

790

-

268

Tissue weight (mg)

5.4

6.3

50 50

-

24.3 -

65

Phospholipid ‘( xgj5 (mg/lOOg) m

Acid extraction

0.003

o+lO3

-

0.060

Total. T; P. hptd (predemin. wt.)

1. THETOTAL WEIGHTOF PHOSPHOLIPID EXTRACTED FROM ENAMEL MATRIX AND DENTINE BEFORE AND AFTER DECALCIFICATION AND SEQUENT ACID EXTRACTION(CALCULATED FROM THE TOTAL AMOUNT OF LIPID PHOSPHORUS RECOVERED)

Enamel Matrix (B)

TABLE

SUB-

0.10

0.13

-

0.42

Total % P. lipid (demin. wt.)

UPON

ENAMEL MATRlX AND DENTINE PHOSPHATIDES

507

TABLE 2. THE DISTRIBUTION OF THE PHOSPHOLIPIDSOF ENAMEL MATRIX % of total lipid phosphorus in each extract

Extract no.

1

2

3

PI

Bph

Let

PE

PS

PA

A

-

16.8 18.3

755 73.9

7.6 7.9

-

-

-

B

-

20.0

65.0

14.4

-

-

20.3

67.3

12.3

-

-

-

A

18.9 19.5

-

-

42.5 48.6

15.8 14.6

8.1 63

14.4 11.0

B

16.2 IO.9

-

8.5 6.4

58*9* 57.4

12.4 12.2

-

4.2 12.9

-

23.8 17.6

-

-

Extraction stage

Undemineralized enamel matrix

Demineralized enamel matrix

Acid extracted t enamel matrix __~_.

8.4

1I.2 _

19.0 25.1 _. ._

49.0 46.1

CL

-

A and B refer to analyses performed on two separate lipid extracts. At least two chromatograms of each extract was analysed for the distribution of lipid phosphorus. * indicates the presence of Lyso PE (8.1 per cent), which is included here together with its parent compound. t Refers to only one series of analyses due to the small amount of lipid.

Phosphorus analysis of chromatograms

qfdentine

In Table 3, the distribution of phosphorus in lipid extracts from dentine is shown. Undemineralized dentine appeared to have a similar phospholipid content as enamel matrix, although some CL and a little lyso Let were seen. The distribution of phosphorus was very similar to that found in undemineralized enamel matrix. Lecithin again occupied 75 per cent of the total extract. The mean total lipid phosphorus was 8.3 pg/g dry undemineralized dentine. Demineralized dentine showed a similar pattern of phosphatides as those extracted from enamel matrix at this stage, although Sph was present (about 15 per cent), whilst CL was absent. The phospholipids PE and PS accounted for over 60 per cent of the total lipid phosphorus present, PI, Sph, and Let making up the bulk of the remainder (in fraction A some PA was present). The mean total lipid phosphorus was 11.6 pg/g dry demineralized tissue. The phospholipids of the acid extract of dentine matrix were very similar to those seen in demineralized extracts, however Sph and CL were absent. Phosphatides PE and PS were present in the largest amounts, PI and PA accounting for the remaining 13 per cent. This extract contained 2-6 rg of phosphorus/g of dry demineralized lipid extracted dentine matrix. Tn all chromatograms obtained from both enamel matrix and dentine, a purplish area (PuX) was seen towards the solvent front when the wet or damp paper was viewed in U.V.light. Although this did not correspond to any known phosphatide and indeed analysis of the area revealed no phosphorus, this material was not an artifact produced

I. M.

508

by the chromatography extracts. TABLE

SHAPIRO,

R. E. W~JTHIERAND

J. T. IRVING

procedures and appeared to be a major constituent of the

3. THE DISTRIBU~ON

OF THE PHOSPHOLIPIDSOF DENTING

y(, of total lipid phosphorus No.

Extraction stage A

1

Undemineralized dentine

3

Demineralized dentine

-.

B

A 2

PI

B

Acid extractedt dentine

10.4 7.6 11.7 IO.6 7.5 6.3

Sph 9.0

Let

PE

PS

PA

9.1 11.3 10.6

77.2* 77.2 67.1% 68*7*

12.5 12.6 20.4 19.2

-

--

14.4 16.2 18.2 16.1

Il.6 12.4 Il.7 18.2

28.7 30.4 35.1 37. I

30.2 30.3 35.2 18.1

4.7 3.2

33.8 30.7 ____

22.7 22.0

4.4 7.5 ___.-____

-

31.5 33.7 __~~~_ ._

CL 1.3 l-3 1.4 1.3 -_--

A and B refer to analyses performed on two separate lipid extracts. At least 2 chromatograms of each extract was analysed for the distribution of lipid phosphorus. * Indicates the presence of a small amount of lyso Let which is here ncluded together with its parent compound. t Refers to only one series of analyses, due to the small amount of 1p .

Distribution of the phosphoiipids

Tables 4 and 5 summarize the distribution of the individual phospholipid expressed as a percentage of the total extracted lipid phosphorus (enamel matrix 52 pg, dentine 39 [Lg). In Table 4 it can be seen that PE and Let were the major phosphatides present in enamel matrix, accounting for 67.2 per cent of the total. The acidic phosphatides were present in smaller though significant amounts. In Table 5, a somewhat similar pattern can be seen for the distribution of the phospholipids of dentine. The non-acidic phosphatides again comprised the bulk of the extracted phospholipids; the acidic phospholipids again made their appearance after demineralization. Although the percentage of lipid extracted after demineralization was less than in the enamel matrix, the percentage of the individual acidic phosphatides in both enamel matrix and dentine corresponded very closely. The level of Let in dentine was nearly double that found in the enamel matrix, whereas the level of PE in dentine was only half that found in the enamel. Nevertheless these two lipids constitute 2/3 to 3/4 of the total lipids found in these tissues. Absorption spectrum of the methanol soluble extract ji-om demineralized enamel matrix

This exhibited an absorption spectrum having a single peak at 280 rnp, characteristic of the presence of protein.

ENAMEL MATRIX AND DENTYNE PHOSPHATIDES

509

TABLE4. THfZDISTRIBWIWN

OF THE INDWIDUAL PHOSPHATIDES OF THE ENAMEL MATRIX (EXPRESSED AS A PERCENTME OF THE TO1 AL PHOSPHORUS EXTRACTED-52 /+) %

-____ Predemineralized

Phosphoiipid _

of total extracted lipid phosphorus Demineralized

Acid extracted

___I-. Total

Sph Lee

8.0 29.0

-2.0

0.9 2.2

8.9 33.2

G PS PA CL

5.0

29.0 6.4 8.0 2.0 6.0 53.47;

0.4 0.8

34.0 68 8.8 2.0 6.0 99.7 y0

42.0 “/, ~__

TOtal

4.3 %

TAHLI:5. THE DISTRIwrwU

01~THE INDIVIDUAL PHOSPHATIDESOF DENTINE (EXPRESSEDAS A PERCENTAGE OF THE TOTAL PHOSPHORUS EXTRACTFD--39 &&g)

7; of Phospholipid --.

Sdan

Predemineralized

total extracted

Demineralized

lipid phosphorus Acid extracted

Sph LeC

7.0 SO.0

4.0 4-o

PE Pl PS PA CL Total

10.0

6.0 3.0 8-O I.0 -

I.5 I.0 I.0 1.o

26.0 7;

60%

1.0 68.0:”

I.5

Total 11.0 56-O

17.5 4.0 9.0 2.0 1.0 lOO*O”/,

black staining of the foetal bovine teeth

Sudanophilia was seen in the enamel matrix of the teeth in all stages of development. In the sections that had been reextracted, there was no loss of sudanophilia when subsequently restained. This was indicative that the sudanophilic material could not be removed by acidified lipid solvents. DISCUSSION

Before demineralization, about 20 mg of phospholipid could be extracted from 100 mg of the foetal bovine dentine. This was a considerably higher amount than was found by DIRKSEN and IKELS (1964) but lower than that found by HESS, LF.E and PECKIIAM (1956). After demineralization with EDTA further amounts of phoxpholipids were made available for extraction, again in greater amounts than found by DIRKSIZN and IKELS (,1964). These differences could be due to (a) the use of foetal hovine dentine (other workers had used human material). or (h) the method ol extraction: particular care was taken here to cnsurc that the lipid solvent pcnetratcd all the dentine particles. This was facilitated by grinding. homogenizing and length) extraction of the tissues

510

1.

M.

SHAPIRO, R.

E.

WIITHIFR AND .I.

T.

IRVING

Prior to demineralization it was found that Let and Sph could be extracted from dentine. This confirmed DIRKSEN’S (1963) findings; however, at this stage some PE was also extracted. In both enamel and dentine Let was easily removed by extraction before demineralization. However, PE had different characteristics. In dentine it was nearly all removed before demineralization of the tissue, whereas in enamel matrix most of the PE was extracted after demineralization. The release of the acidic phosphatides (PI, CL, PS and PA) was not seen until after demineralization. In both cases, acid extraction yielded only a small percentage of the total extractable lipid phosphorus; PE and Let being the principal phosphatides involved. An unknown compound “PuX" appeared in all the chromatograms of enamel and dentine and would appear to correspond to a similar unknown reported by DIRKSEN (1963) using a one dimensional system. It was possible that this chromatographic spot was an artifact produced by contamination from silica-gel, silicone grease, chromatographic solvents or EDTA. However, when these materials were examined by paper chromatography, no corresponding component was seen. The unknown contained no phosphorus, choline (negative reaction to Dragendorf reagent) or free amino groups (negative reaction to Ninhydrin). Its characterization is now being investigated more fully and will be described in a later communication. The presence of a relatively high amount of phospholipid in enamel matrix compared with dentine is believed to be the first report of this kind. The greatest amount of phosphatide was liberated after the tissue was demineralized, IO times more lipid being released from enamel matrix than dentine. It is interesting to note that in both tissues investigated a similar spectrum of phospholipids was removed at each extraction stage, although the amount and proportion of each component was different. It may be concluded that in both enamel matrix and dentine (a) lipid extraction before demineralization removes primarily the neutral phosphatides, in this case mainly Lee but with some Sph and PE. (b) a number of the phospholipids can only be removed after demineralization of the tissues. These tend to be acidic phospholipids, many of which have been shown to exhibit a strong af?inity for calcium (WOOLEY and CAMBELL. 1962; KIMIZUKI and KOKETSU, 1962) and secondarily for phosphate (BADER, 1962, 1964). It is possibly that these lipids play some part in the calcification mechanism by their interaction with these mineral ions. (c) a small amount of tightly bound phospholipid is present which can only be removed by treatment with mild acid solvents after demineralization. These lipids are probably more strongly associated with proteins and cannot be extracted until the linkage is broken. The presence of a methanol soluble protein from demineralized enamel matrix indicates that it is highly probable that the acidic phosphatides are in some sort of association with protein or peptides of the enamel (as well as calcium or phosphorus) and form lipoprotein complexes. The nature of this protein is being further studied and will be reported elsewhere. The appearances of sudanophilia in bovine foetal teeth is in agreement with IRVING (1963). Further extraction and subsequent re-staining of the tissues failed to remove the sudanophilia from either enamel or dentine. Thus it is probable that this material was not removed by the extraction procedures employed to remove the lipids from the

ENAMEL

MATRIXANDDENTlUrPHOSPHATIDl!.s

511

tissues. It is interesting to note that acid extraction completely removed the sudanophilia lrom both calcifying cartilage and bone. Acknowledgements-This project was supported by U.S. Public Health Grant No. D 876, National Institute of Dental Research. I. M. Shapiro is in receipt of a Nutlield Foundation Dental Research Fellowship. R&sum&-Les phospholipides de la matrice d’email et de dentine de foetus bovins, extraits avant et apres dtminbalisation, sont analyses. La matrice de I’Cmail contient un pourcentage nettement plus 61eve de lipide que la dentine. Avant dtmintralisation, settlement 40% des phospholipides totaux ont pu etre extraits de la matrice de l’email, alors que 2/3 du total ont pu etre extraits de la dentine. Dans les deux tissus, les phosphatides neutres ont Ctt extraits surtout avant dknintralisation, alors que les phosphatides acidiques ont CtC extraits settlement apres dtmineralisation. A chaque stade d’extraction, les phosphatides lib&s de P&mail et de la dentine sont identiques qualitativement, mais different en quantites et proportions. II semble que les phosphatides acides puissent intervenir dans le processus de la calcification. Zusammenfassung-In der fetalen Schmelzmatrix und im Dentin von Rindern wurden die Phospholipide untersucht, die vor und nach der Mineralisation extrahiert worden waren. Die Schmelzmatrix enthielt einen vie1 hiiheren Prozentsatz Lipide als Dentin. Vor der Demineralisation konnte lediglich 40% des gesamten Phospholipids aus der Schmelzmatrix extrahiert werden, wihrend iiber 2/3 des Gesamten aus dem Dentin gewonnen werden konnte. In beiden Geweben wurden neutrale Phosphatide primlr vor der Demineralisation gewonnen, wohingegen saure Phosphatide nur nach der Demineralisation extrahiert wurden. Bei jedem Extraktionsschritt waren die aus der Schmelzmatrix und dem Dentin befreiten Phosphatide qualitativ Ihnlich, sie unterschieden sich jedoch in ihren Mengen und Proportionen. Es wird angenommen, dass die sauren Phosphatide eine Rolle im Verkalkungsprozess spielen dtirften. REFERENCES AUAUAT~U,K. 1929. Das Vorkommon von Fett in Schmehz des Zahnes. Vrtf&+rr. F. Z&h 45, 33-47. ALLRED,H. 1964. Histochemical observations of dentine lipids (abstr.) J. den/. Res. 43, 971. BADER, H. 1962. The uptake of inorganic phosphorus by the lipid extract of rat liver. Biochem. biophys. Acta 65, 178-180. BADER,H. 1964. I)ber das Bindings-vermogen der lipide fur an-organisches phosphat. Biopllwih1, 370-372. CREUSS,R. L. and CLARK, I. 1965. Alterations in the lipids of bone by hyper vitaminosis A and D. Biochem J. %,262-265. DIRKSEN,T. R. 1963. Lipid components of sound and carious dentin. J. dent. Res. 42, 128-32. DIRKSEN,T. R. and IKELS, K. G. 1964. Quantitative determination of some constituent lipids in human dentin. J. dent. Res. 43, 246-251. HESS, W. C., LEE, C. Y. and PECKHAM,S. C. 1956. The lipid content of enamel and dentin, J. dent. Res. 35, 273-275. HOSOYA,N., WATANABE,T. and FUJIUORI,A. 1964. The action of vitamin D in vivo and in vitro on the incorporation of DL_(3 -14C) serine into phosphatidylserine. Biochem. bioph,vs. Acra 84, 770-771. IRVING, J. T. 1958. A histological stain for newly calcified tissues, Nature, Lond. 181, 704705. IRVING, J. T. 1963. Calcification of the organic matrix of enamel, Arch. oral Biol. 8. 773-774. IRVING,J. T. 1963. The sudanophil material at sites of calcification, Arch. oral Biol.’ 8, 735-743. JOHNSON,L. C. 1960. Calcification in Bioloaieaf Svstems. v. 117. No. 64 American Association for the ‘. Advancement of Science, Washington. . K~MIZUKI,H. and KOKETSU,K. 1962. Binding of calcium ion to lecithin film. Natrrre, Lond. 196, 995-996.

I. M. SHAPIRO,R. E. WIJ~HIERAND J. T. IRVING

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KRASNOW,F. 1934. Cholesterol and lecithin in teeth and saliva. J. dent. Res. 30, 837-839. LEVIN,P. T., GLIMCHER,M. J. and B~NAR, I. C. 1964. Collagenous layer covering the crown enamel

of unerupted

permanent

human teeth.

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