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Fatty acid composition of neutral lipids and phospholipids of enamel and dentine from human molars
Archs.oral Biol.Vol. 19, pp.293 to 298. PergamonPress 1974. Printedin Great Britain
FATTY ACID COMPOSITION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS OF ENAMEL AND DENTINE FROM HUMAN MOLARS R. E. S. F’ROUT and A. A.
ODUTUCA
The Department of Biochemistry, University of Sheffield,Sheffield,SlO 2TN, England
Summary-Neutral lipids and phospholipids were extracted from enamel and dentine of human molars, hydrolysed and the fatty acid composition of cholesterol esters, triglycerides, free fatty acids plus mono- and di-glycerides and total neutral lipids were determined. Lecithin, sphingomyelin plus phosphatidyl serine and phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidic acid plus cardiolipin and total phospholipid fatty acids were also analysed. The major fatty acids present were palmitic, stearic and oleic, palmitoleic and linoleic acids being major components in some phospholipids. The relative proportions of the fatty acids from both neutral and phospholipids were similar in dentine and enamel. Both qualitative and quantitative analyses were similar to those obtained from normal rat teeth.
lNTRODUCl’ION
Analyses of the fatty acids present in lipids extracted from dental tissues of various animals have been reported by a number of workers; bovine dental pulp (Graziano, 1962), molars of 16 different animal species (Das and Harris, 1970a), fossil teeth (Das and Harris, 1970b), whole human dentine (Rabinowitz et al., 1967), human root dentine and enamel (Prout and Shutt, 1970a) and rat enamel and dentine (Prout and Shutt, 1970b; Prout and Atkin, 1973). The fatty acids of the major neutral lipid components of calf enamel matrix have been reported by Fincham, Burkland and Shapiro (1972). Some analyses of the fatty acid composition of individual lipid classes extracted from various oral and calcified tissues have also been carried out on pig gingiva (Rabinowitz, Bailey and Marsh, 1971), bovine bone (Shapiro, 1971), human bone (Dirksen and Marinetti, 1970) and calf enamel (Fincham et al., 1972). Comprehensive analyses of the fatty acid composition of individual neutral lipids and individual phospholipids of rat enamel and dentine have been carried out by Odutuga and Prout (1973). No analyses, however, appear to have been made on the fatty acid composition of the neutral lipids and phospholipids of human enamel and dentine. MATERIALS
AND METHODS
Freshly extracted caries-free human molars were cleaned free of surrounding soft tissue and bone and were kept deep frozen until use. The teeth were cut in half, lengthways, and the contents of the pulp cavity removed using a water-cooled high-speed dental drill. The teeth were then washed with distilled water, freezedried and pulverized in a stainless steel ball-mill to pass through a 240 mesh sieve. Pure enamel and pure den-
tine samples were obtained by differential flotation with aqueous cadmium tungstoborate solution (Prout and Shutt, 197Oc). The samples were washed with water and divided into four groups: (1) 0.4 g of enamel; (2) 1.0 g of dentine: (3) 1.8 g of enamel; (4) 4.5 g of dentine. Two separate analyses, with the same analytical procedures were carried out. In a pilot analysis, groups (1) and (2) were used and in the second analysis groups (3) and (4). The procedures for obtaining lipid extracts before and after decalcification were as described by Prouf Odutuga and Tring (1973). The lipids extracted before and after decalcification were combined. The total lipid extracts from each of the four groups were separated into neutral lipids and phospholipids and analysed qualitatively and quantitatively as described previously (Odutuga and Prout, 1973). Fatty acid methyl esters were analysed by gas-liquid chromatography with a Pye 104 Chromatograph (W. G. Pye and Co. Ltd., Cambridge, England) using a 5-ft glass column of 10 per cent poly(diethyleneglyco1) adipate on 100/12Omesh Celite washed with 3 per cent phosphoric acid. The methyl esters were separated by temperature programming in a stream of nitrogen with a flow-rate of 40 ml/mm and were identified by comparing retention times with those of a standard mixture of methyl esters and from a plot of the logarithm of the retention time against carbon number and degree of unsaturation. The area of each peak obtained from the recorder was measured by triangulation. The response of the Chromatograph was calibrated with an equimolar standard mixture of methyl esters of caprylit, capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, behenic and docosahexaenoic acids (Sigma Chemical Co. Ltd., London).
293
R. E. S. PROUT
294
and A. A. ODUTUGA
Table 1. Fatty acid analyses of neutral lipids and phospholipids of sound human dentine and enamel Pulverized wt of tissue (mg)
Total lipid (mg)
Den tine Lipid content (mg) Fatty acids (mg) Fatty acids % of lipid
1000
34
Enamel Lipid content (mg) Fatty acids (mg) Fatty acids % of lipid
400
Tissue
RESULTS
Dentine and enamel contain 0.34 and 0.53 per cent lipid respectively. The amount of neutral and phospholipid present in these tissues are given in Tables 1 and 2. The qualitative analyses of the fatty acids in total neutral lipids and total phospholipids in the first ex-
.Total neutral lipids (mg)
Total phospholipids (mg)
2.24 1.05 46-9
1.16 0.75 64.7
152 0.76 500
0.60 0.38 63.3
2.12
periment are shown in Table 1. The total neutral lipids and the total phospholipids from dentine contained 46.9 and 64.7 per cent fatty acids respectively, whilst fatty acids were found to constitute 50.0 per cent of the total neutral lipids and 63.3 per cent of the total phospholipids from enamel. In the second experiment (Table 2), fatty acids as a percentage of total neutral or phospholipid in the different tissues, calculated by addition of fatty acids of Chole&rol esters
Trlglytides r-l
s’
Free fatty acids t monoglyceridest diglymidea
E
2
soy
g $ 0
r
SO
Free fatty acids t monoglycaides + diglycefides 5o
Total neutral lipids
Fig. 1. Histogram of the fatty acid composition of neutral lipids extracted from human molar dentine. Fatty acid composition isexpressedaspercentage totalfattyacid. Particular fatty acids are indicated by carbon number.
Fotty ocld
Fig. 2. Histogram of fatty acid composition of neutral lipids extracted from human molar enamel.
1800
Enamel Lipid content (mg) Fatty acids (mg) Fatty acid % of lipid Theoretical fatty acids % of lipid 9.54
15.0
Total lipid (mg)
38.9
88.2
1.94 1.70 87.6
88-9
2.59 2.28 88.0
PS = Phosphatidyl
1.83 0.71 38.8
39,6
2.79 -
2.75 1.13 41.1
51.6
6.97 3.43 49.2
47,2
9.94 4.56 45-9
Total neutral lipid
66.1
1.14 0.73 64.0
66.5
2.16 1.34 62.0
Lecithin
serine; PI = Phosphatidyl
78.0
0.41 031 75.6
78.6
0.50 0.38 76.0
Neutral lipids FFA + monoTri+dichol. glycglycest. erides erides
4.1 -
Chol.
FFA = Free fatty acids; SPH = Sphingomyelin; PA = Phosphatidic acid; Card = Cardiolipin.
4500
Dentine Lipid content (mg) Fatty acids (mg) Fatty acid % of lipid Theoretical fatty acids % of lipid
Tissue
Pulverized wt of tissue (mg)
70.7
0.56 0.39 69.6
70.5
1.04 0.70 67.3
PE
75.4
0.36 0.27 75.0
74.8
0.74 0.49 66,2
PA+ Card.
inositol; PE = Phosphatidyl
57.2
0.51 0.29 56.9
56.5
1.12 0.62 55-4
SPH + ps+ PI
Phospholipids
Table 2. Fatty acid analyses of neutral lipids and phospholipids of sound human dentine and enamel
ethanolamine;
62.3
2.57 1.61 62.6
65.1
5.06 3.14 62.1
Total phospholipids
R. E. S. Pnour
296
50
r
Sphingomyelin+ phosphotidyl serine + phosphatidyl inositol
:‘/: Phasphatldic acid + cardiolipin
2:: Total phospholipids
and A. A. OIXWUGA
Palmitic, stearic and oleic acids were also the principal fatty acids of the phospholipids from both dentine and enamel (Figs. 3 and 4). Many of the phospholipids also contain relatively high proportions of linoleic and linolenic (C18:3) acid. The sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol fraction of the phospholipids also contains a relatively high proportion of palmitoleic acid (C16: 1). Trace amounts of arachidonic acid were found in the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol and phosphatidic acid plus cardiolipin fractions from dentine and in the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol fraction from enamel. Total neutral lipids from dentine and enamel contained 39 and 31 per cent of palmitic acid, 20 and 25 per cent of stearic acid and 20 per cent of oleic acid respectively. Low concentrations of myristic (C14:0), linoleic and linolenic acids were also present in the neutral lipids from both tissues. Neutral lipids from enamel, however, contained slightly higher amounts of linoleic and linolenic acids than the neutral lipids from dentine. Total phospholipids from both dentine and enamel contained palmitic acid 28 and 22 per cent, stearic acid 23 and 21 per cent and oleic acid 23 and 19 per cent. !L,
Fotty
Lecithi,.m,
acid
Fig. 3. Histogram offattyacid composition of phospholipids extracted from human molar dentine.
the individual lipid classes, gave essentially the same results as those in the first experiment. In Table 2, the theoretical percentage of fatty acid present in each neutral and phospholipid class and in total neutral and phospholipids has been calculated. The mean molecular weight of each fatty acid in each neutral and phospholipid class was calculated from the proportions of the different constituent fatty acids shown in Figs. 14. The percentage fatty acid in total neutral lipid and phospholipid was calculated from the percentage fatty acid in the different lipid classes taking into account the relative proportions of different lipids making up the total neutral and phospholipids in the different tissues (Odutuga and Prout, 1973b). Figures 1 and 2 show histograms of the fatty acid composition of neutral lipids of dentine and enamel, respectively. The major fatty acid components were palmitic acid (C16:0), stearic acid (C18:O) and oleic acid (C18: 1). Small concentrations of linoleic acid (C18:2) were also present in all the neutral lipids except cholesterol esters. Trace amounts of arachidonic acid (C20:4) were found in the free fatty acids plus monoglycerides plus diglycerides fraction.
50
Sphingamyelin+phosphotidyl serine + phwphatldyl inositoi
Phosphatidyl ethanolamine
Ptvxphatidlc ocld
l
cardiolipin
Fig. 4. Histogram of fattyacidcomposition of phospholipids extracted from human molar enamel.
Fatty acid composition
of lipids from human
respectively. On the whole, enamel contained higher concentrations of linoleic acid than dentine, the greater part of which was found in phospholipids. DISCUSSION
Human dentine contains 0.18 per cent lipid extracted from demineralized tissue (Dirksen and Ikels, 1964). Fincham et al. (1972) found that calf enamel contained 0.11 per cent neutral lipids representing 65 per cent of the total lipids extracted from this tissue. Rat enamel and dentine contain 0.57 and 0.35 per cent lipid respectively (Prout et al., 1973) and human enamel and dentine contain 051 and 0.33 per cent lipid respectively (Odutuga and Prout, 1974); these values are comparable to the values of 060 and 0.36 per cent obtained for human enamel and dentine respectively by Hess, Lee and Peckham (1956). In the present study, neutral lipids were found to represent 66 and 72 per cent of the total lipids extracted from dentine and enamel respectively. These values agree with the values of 64-66 and 72-75 per cent obtained for rat dentine and enamel respectively (Odutuga and Prout, 1973). The percentage of fatty acid in the total neutral lipids is lower than the percentage in the total phospholipids (Tables 1 and 2) owing to the inclusion of cholesterol in the total neutral lipids. In Table 2, the experimental recovery of fatty acids from the various lipids calculated as per cent of lipid is over 90 per cent in all cases when compared to the theoretical fatty acid content. The amount of fatty acid present in the teeth was calculated from the results to be 283 mg/lOO g of enamel 176 mg/lOO g of dentine. Approximately 50 per cent of total tissue lipid is fatty acid (Table 2) therefore from the above figures the lipid content of enamel should be 283 x 2 = 566 mgjlO0 g. This is similar to the figure of 571 mg/lOO g of lipid obtained from rat enamel (Odutuga and Prout, 1973) and to the figure of 530mg/lOOg of lipid extracted from human enamel (calculated from Tables 1 and 2). Similarly, the lipid content of dentine is calculated to be 352 mgjlO0 g; this also agrees with the figure of 357 mg/100 g of lipid obtained for rat dentine (Odutuga and Prout, 1973),and with the figure of 336 g/100 g of lipid extracted from human dentine (Tables 1 and 2). These figures are in the same range as the values of 510 mg/lOOg and 570 mg/lOO g of lipid extracted from human and rat enamel respectively, 330 mg/lOO g and 350 mg/lOO g of lipid extracted from human and rat dentine respectively. which have been reported previously (Prout et al., 1973; Odutuga and Prout, 1973). The difference in fatty acid composition of enamel and dentine becomes more significant when the amount of fatty acid present in the organic residue is calculated, as distinct from the total calcified tissue, since enamel contains only approximately 1.2 per cent organic matrix and dentine contains approximately 20 per cent (Jenkins, 1970). Enamel organic matrix contained approximately 23.6 g of fatty acid per 100 g, whilst dentine organic
molars
291
matrix contained 880 mg of fatty acid per 100 g. As 50 per cent of lipid in enamel and dentine is fatty acid, (23.6 x 2) = 47.2 per cent lipid is present in human enamel organic matrix and (880 x 2) 1.76 per cent lipid is present in dentine organic matrix. These figures agree closely with the percentage lipid content of 42.5 and 1.65 per cent reported previously in human enamel and dentine (Odutuga and Prout, 1973) and in rat dentine (Prout et al, 1973). The value for enamel, however, is higher than the 35.0 per cent found in rat enamel by Prout et al. (1973); this difference is not considered to be significant in view of the fact that the value for the organic matrix of rat enamel is different from that of human, rat enamel containing 1.63 per cent, and human enamel coqtaining 1.2 per cent by weight (Jenkins, 1970; Prout et al., 1973). From the results obtained in the present study, it was calculated that enamel matrix contained a concentration of fatty acid 27 times greater than that of the organic matrix of dentine. This agrees with the value of 26 times more lipid reported previously (Odutuga and Prout, 1974) and is similar to the value of 20 times more lipid reported for the rat (Prout et al., 1973). The present data provide a detailed comparison of the fatty acid composition of different lipids in the enamel and dentine of human molars. The fatty acid distribution is similar to that previously reported in total human enamel and dentine (Rabinowitz et al., 1967; Prout and Shutt, 197Oa),in total rat enamel and dentine and neutral !ipid and phospholipid of rat enamel and dentine (Prout and Shutt, 1970b; Prout and Atkin, 1973; Odutuga and Prout, 1973) and, in other hard tissues (Das and Harris, 1970a, b; Fincham et al., 1972), the major fatty acids being palmitic, stearic and oleic acid. However, these results are substantially different from the analyses of bovine bone (Shapiro, 1971) where palmitic and stearic acids were noted in smaller concentrations than were found in the present study. It has been observed in the present study that enamel contained more linoleic and linolenic acids than dentine; the greater part of these fatty acids was found in the phospholipids (mostly in the acidic phospholipids). A similar observation was made in the rat (Odutuga and Prout, 1973). In the present study trace amounts of arachidonic acid were detected in the neutral and phospholipids from human molars in contrast to the rat where the free fatty acid plus monoglycerides plus diglycerides neutral lipid fraction of incisor dentine and enamel contains 15.0 per cent arachidonic acid and the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol phospholi$d fraction of molar dentine and the phosphatidyl ethanolamine of incisor enamel contain 15.0 per cent and 7.0 per cent respectively (Odutuga and Prout, 1973). Acknowledgements-We thank Mr. P. Hancock for skilled technical assistance and the Nigerian Government for provision of a support grant for A. A. Odutuga. The work forms part of a thesis to be submitted for the degree of Ph. D. by A.A.O.
298
R. E. S. PROUT and A. A. ODUTUGA REFERENCES
Das S. K. and Harris R. S. 1970a. Fatty acids in the tooth lipids of 16 animal species. J. dent. Res. 49, 119-125. Das S. K. and Harris R. S. 1970b. Lipids and fatty acids in fossil teeth. J. dent. Res. 49, 126-130. Dirksen T. R. and Ikels K. G. 1964. Quantitative determination of some constituent lipids in human dentine. J. dent. Res. 43,246-251. Dirksen T. R. and Marinetti G. V. 1970. Lipids of bovine enamel and dentin and human bone. Calc. Zss. Res. 6, I10. Fincham A. G., Burkland G. A. and Shapiro I. M. 1972. Lipophilia of enamel matrix. A chemical investigation of the neutral lipids and lipophilic proteins of enamel. Calc. 7iss. Res. 9. 247-259. Graziano V. 1962. Determinazione gas chromatographica degli acidi grassi nella polpa di denti di bue. Boll. sot. ital. Biol. sper. 38, 13041306. Hess W. C.. Lee C. Y. and Peckham S. C. 1956. The lipid content of enamel and dentin. J. denr. Res. 35, 273-275. Jenkins G. N. 1970. The Physiology of’ the Mouth, 3rd Edi. Blackwell, Oxford. Odutuga A. A. and Prout R. E. S. 1973. Fatty acid composition of neutral lipid and phospholipids of enamel and dentine from rat incisors and molars. Archs oral Biol. 18, 689497.
Odutuga A. A. and Prout R. E. S. 1974. Lipid analysis of human enamel and dentine. Archs oral Biol. 19, in press. Prout R. E. S.. Odutuga A. A. and Tring F. C. 1973. Lipid analyses of rat enamel and dentine. Archs oral Biol. 18, 373-380. Prout R. E. S. and Atkin E. R. 1973. Effect of diet deficient in essential fatty acid on fatty acid composition of enamel and dentine of the rat. Archs oral Biol. 18, 583-590. Prout R. E. S. and Shutt E. R. 1970a. Analysis of fatty acids in human root dentine and enamel. Archs oral Biol. 15, 28 l-286. Prout R. E. S. and Shutt E. R. 1970b. Analysis of fatty acids in rat enamel and dentine. Archs oral Biol. 15, 1105-l 106. Prout R. E. S. and Shutt E. R. 197Oc. Separation of enamel and dentine using cadmium tungstoborate solution. Archs oral Biol. l&559-561. Rabinowitz J. L., Bailey T. A. and Marsh J. B. 1971. Polar and neutral lipids of pig gingiva. Archs oral Biol. 16, 1195.-1205. Rabinowitz J. L., Luddy F. E.. Barford R. A., Herb S. F., Orlean S. L. and Cohen D. W. 1967. Lipid determination in powdered human dentin by thin-layer and gas-liquid chromatography. J. dent. Res. 46, 1086-1089. Shapiro I. M. 1971. The neutral lipids of bovine bone. Archs oral Biol. 16,411421.
Rbsum&Les
lipides Heutres et les phospholipides ont ktk extraites de l’kmail et de la dentine de molaires humaines, hydrolyskes et on a dktermink la composition des acides gras des esters du cholestkrol, des triglyckides, les acides.gras libres plus les mono- et les di-glyckrides et les lipides neutres totales. On a analysk aussi la ltcithine, la sphingomyirline plus la phosphatidyle sCrine plus l’inositol phosphatidyle, Ethanolamine phosphatidyle, l’acide phosphatidique plus la cardiolipine et les acides gras phospholipidiques totaux. Les acides gras majeurs prksents ktaient les acides palmitique, stkarique et okique, palmitoltique et linokique, Ctant les constituantes majeures dans quelques phospholipides. Les proportions relatives des acides gras des lipides neutres et phospholipides ont CtC similaires dans la dentine et l’kmail. Les analyses qualitatives et quantitatives Ctaient similaires a celles obtenues des dents des rats normaux.
ZusammenfassungpEs
sind neutrale Lipide und Phospholipide aus Schmelz und Dentin von menschlichen Molaren ausgeschieden und hydrolisiert worden, und es wurden die Fettlurenzusammenstellung der Cholesterolesther, Triglyceriden, freie Fettsiiuren plus Mono- und Diglyceriden und gesamte neutrale Lipiden ermittelt. Auch sind Lezithin, Sphingomyeline plus Phosphatidylserin und Phosphatidylinosit, Phosphatidytithanolamin, Phosphatidslure plus Kardiolipine und gesamte Phospholipidefettskren analysiert worden. Die vorhandenen hauptG&lichen Fettsluren waren Palmitin, Trlnen und tjlkiuren und Palmitolein und LinolQuren waren Hauptbestandteile in einigen Phospholipiden. Die relativen Proportionen der Fetttiuren sowohl von neutralen Stoffen wie von Phospholipiden waren im Dentin und Schmelz Phnlich. Sowohl qualitative wie quantitative Analysen lhnelten denen, die von normalen Rattenzghnen erhalten wurden.
FATTY ACID COMPOSITION OF NEUTRAL LIPIDS AND PHOSPHOLIPIDS OF ENAMEL AND DENTINE FROM HUMAN MOLARS R. E. S. F’ROUT and A. A.
ODUTUCA
The Department of Biochemistry, University of Sheffield,Sheffield,SlO 2TN, England
Summary-Neutral lipids and phospholipids were extracted from enamel and dentine of human molars, hydrolysed and the fatty acid composition of cholesterol esters, triglycerides, free fatty acids plus mono- and di-glycerides and total neutral lipids were determined. Lecithin, sphingomyelin plus phosphatidyl serine and phosphatidyl inositol, phosphatidyl ethanolamine, phosphatidic acid plus cardiolipin and total phospholipid fatty acids were also analysed. The major fatty acids present were palmitic, stearic and oleic, palmitoleic and linoleic acids being major components in some phospholipids. The relative proportions of the fatty acids from both neutral and phospholipids were similar in dentine and enamel. Both qualitative and quantitative analyses were similar to those obtained from normal rat teeth.
lNTRODUCl’ION
Analyses of the fatty acids present in lipids extracted from dental tissues of various animals have been reported by a number of workers; bovine dental pulp (Graziano, 1962), molars of 16 different animal species (Das and Harris, 1970a), fossil teeth (Das and Harris, 1970b), whole human dentine (Rabinowitz et al., 1967), human root dentine and enamel (Prout and Shutt, 1970a) and rat enamel and dentine (Prout and Shutt, 1970b; Prout and Atkin, 1973). The fatty acids of the major neutral lipid components of calf enamel matrix have been reported by Fincham, Burkland and Shapiro (1972). Some analyses of the fatty acid composition of individual lipid classes extracted from various oral and calcified tissues have also been carried out on pig gingiva (Rabinowitz, Bailey and Marsh, 1971), bovine bone (Shapiro, 1971), human bone (Dirksen and Marinetti, 1970) and calf enamel (Fincham et al., 1972). Comprehensive analyses of the fatty acid composition of individual neutral lipids and individual phospholipids of rat enamel and dentine have been carried out by Odutuga and Prout (1973). No analyses, however, appear to have been made on the fatty acid composition of the neutral lipids and phospholipids of human enamel and dentine. MATERIALS
AND METHODS
Freshly extracted caries-free human molars were cleaned free of surrounding soft tissue and bone and were kept deep frozen until use. The teeth were cut in half, lengthways, and the contents of the pulp cavity removed using a water-cooled high-speed dental drill. The teeth were then washed with distilled water, freezedried and pulverized in a stainless steel ball-mill to pass through a 240 mesh sieve. Pure enamel and pure den-
tine samples were obtained by differential flotation with aqueous cadmium tungstoborate solution (Prout and Shutt, 197Oc). The samples were washed with water and divided into four groups: (1) 0.4 g of enamel; (2) 1.0 g of dentine: (3) 1.8 g of enamel; (4) 4.5 g of dentine. Two separate analyses, with the same analytical procedures were carried out. In a pilot analysis, groups (1) and (2) were used and in the second analysis groups (3) and (4). The procedures for obtaining lipid extracts before and after decalcification were as described by Prouf Odutuga and Tring (1973). The lipids extracted before and after decalcification were combined. The total lipid extracts from each of the four groups were separated into neutral lipids and phospholipids and analysed qualitatively and quantitatively as described previously (Odutuga and Prout, 1973). Fatty acid methyl esters were analysed by gas-liquid chromatography with a Pye 104 Chromatograph (W. G. Pye and Co. Ltd., Cambridge, England) using a 5-ft glass column of 10 per cent poly(diethyleneglyco1) adipate on 100/12Omesh Celite washed with 3 per cent phosphoric acid. The methyl esters were separated by temperature programming in a stream of nitrogen with a flow-rate of 40 ml/mm and were identified by comparing retention times with those of a standard mixture of methyl esters and from a plot of the logarithm of the retention time against carbon number and degree of unsaturation. The area of each peak obtained from the recorder was measured by triangulation. The response of the Chromatograph was calibrated with an equimolar standard mixture of methyl esters of caprylit, capric, lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, arachidonic, behenic and docosahexaenoic acids (Sigma Chemical Co. Ltd., London).
293
R. E. S. PROUT
294
and A. A. ODUTUGA
Table 1. Fatty acid analyses of neutral lipids and phospholipids of sound human dentine and enamel Pulverized wt of tissue (mg)
Total lipid (mg)
Den tine Lipid content (mg) Fatty acids (mg) Fatty acids % of lipid
1000
34
Enamel Lipid content (mg) Fatty acids (mg) Fatty acids % of lipid
400
Tissue
RESULTS
Dentine and enamel contain 0.34 and 0.53 per cent lipid respectively. The amount of neutral and phospholipid present in these tissues are given in Tables 1 and 2. The qualitative analyses of the fatty acids in total neutral lipids and total phospholipids in the first ex-
.Total neutral lipids (mg)
Total phospholipids (mg)
2.24 1.05 46-9
1.16 0.75 64.7
152 0.76 500
0.60 0.38 63.3
2.12
periment are shown in Table 1. The total neutral lipids and the total phospholipids from dentine contained 46.9 and 64.7 per cent fatty acids respectively, whilst fatty acids were found to constitute 50.0 per cent of the total neutral lipids and 63.3 per cent of the total phospholipids from enamel. In the second experiment (Table 2), fatty acids as a percentage of total neutral or phospholipid in the different tissues, calculated by addition of fatty acids of Chole&rol esters
Trlglytides r-l
s’
Free fatty acids t monoglyceridest diglymidea
E
2
soy
g $ 0
r
SO
Free fatty acids t monoglycaides + diglycefides 5o
Total neutral lipids
Fig. 1. Histogram of the fatty acid composition of neutral lipids extracted from human molar dentine. Fatty acid composition isexpressedaspercentage totalfattyacid. Particular fatty acids are indicated by carbon number.
Fotty ocld
Fig. 2. Histogram of fatty acid composition of neutral lipids extracted from human molar enamel.
1800
Enamel Lipid content (mg) Fatty acids (mg) Fatty acid % of lipid Theoretical fatty acids % of lipid 9.54
15.0
Total lipid (mg)
38.9
88.2
1.94 1.70 87.6
88-9
2.59 2.28 88.0
PS = Phosphatidyl
1.83 0.71 38.8
39,6
2.79 -
2.75 1.13 41.1
51.6
6.97 3.43 49.2
47,2
9.94 4.56 45-9
Total neutral lipid
66.1
1.14 0.73 64.0
66.5
2.16 1.34 62.0
Lecithin
serine; PI = Phosphatidyl
78.0
0.41 031 75.6
78.6
0.50 0.38 76.0
Neutral lipids FFA + monoTri+dichol. glycglycest. erides erides
4.1 -
Chol.
FFA = Free fatty acids; SPH = Sphingomyelin; PA = Phosphatidic acid; Card = Cardiolipin.
4500
Dentine Lipid content (mg) Fatty acids (mg) Fatty acid % of lipid Theoretical fatty acids % of lipid
Tissue
Pulverized wt of tissue (mg)
70.7
0.56 0.39 69.6
70.5
1.04 0.70 67.3
PE
75.4
0.36 0.27 75.0
74.8
0.74 0.49 66,2
PA+ Card.
inositol; PE = Phosphatidyl
57.2
0.51 0.29 56.9
56.5
1.12 0.62 55-4
SPH + ps+ PI
Phospholipids
Table 2. Fatty acid analyses of neutral lipids and phospholipids of sound human dentine and enamel
ethanolamine;
62.3
2.57 1.61 62.6
65.1
5.06 3.14 62.1
Total phospholipids
R. E. S. Pnour
296
50
r
Sphingomyelin+ phosphotidyl serine + phosphatidyl inositol
:‘/: Phasphatldic acid + cardiolipin
2:: Total phospholipids
and A. A. OIXWUGA
Palmitic, stearic and oleic acids were also the principal fatty acids of the phospholipids from both dentine and enamel (Figs. 3 and 4). Many of the phospholipids also contain relatively high proportions of linoleic and linolenic (C18:3) acid. The sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol fraction of the phospholipids also contains a relatively high proportion of palmitoleic acid (C16: 1). Trace amounts of arachidonic acid were found in the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol and phosphatidic acid plus cardiolipin fractions from dentine and in the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol fraction from enamel. Total neutral lipids from dentine and enamel contained 39 and 31 per cent of palmitic acid, 20 and 25 per cent of stearic acid and 20 per cent of oleic acid respectively. Low concentrations of myristic (C14:0), linoleic and linolenic acids were also present in the neutral lipids from both tissues. Neutral lipids from enamel, however, contained slightly higher amounts of linoleic and linolenic acids than the neutral lipids from dentine. Total phospholipids from both dentine and enamel contained palmitic acid 28 and 22 per cent, stearic acid 23 and 21 per cent and oleic acid 23 and 19 per cent. !L,
Fotty
Lecithi,.m,
acid
Fig. 3. Histogram offattyacid composition of phospholipids extracted from human molar dentine.
the individual lipid classes, gave essentially the same results as those in the first experiment. In Table 2, the theoretical percentage of fatty acid present in each neutral and phospholipid class and in total neutral and phospholipids has been calculated. The mean molecular weight of each fatty acid in each neutral and phospholipid class was calculated from the proportions of the different constituent fatty acids shown in Figs. 14. The percentage fatty acid in total neutral lipid and phospholipid was calculated from the percentage fatty acid in the different lipid classes taking into account the relative proportions of different lipids making up the total neutral and phospholipids in the different tissues (Odutuga and Prout, 1973b). Figures 1 and 2 show histograms of the fatty acid composition of neutral lipids of dentine and enamel, respectively. The major fatty acid components were palmitic acid (C16:0), stearic acid (C18:O) and oleic acid (C18: 1). Small concentrations of linoleic acid (C18:2) were also present in all the neutral lipids except cholesterol esters. Trace amounts of arachidonic acid (C20:4) were found in the free fatty acids plus monoglycerides plus diglycerides fraction.
50
Sphingamyelin+phosphotidyl serine + phwphatldyl inositoi
Phosphatidyl ethanolamine
Ptvxphatidlc ocld
l
cardiolipin
Fig. 4. Histogram of fattyacidcomposition of phospholipids extracted from human molar enamel.
Fatty acid composition
of lipids from human
respectively. On the whole, enamel contained higher concentrations of linoleic acid than dentine, the greater part of which was found in phospholipids. DISCUSSION
Human dentine contains 0.18 per cent lipid extracted from demineralized tissue (Dirksen and Ikels, 1964). Fincham et al. (1972) found that calf enamel contained 0.11 per cent neutral lipids representing 65 per cent of the total lipids extracted from this tissue. Rat enamel and dentine contain 0.57 and 0.35 per cent lipid respectively (Prout et al., 1973) and human enamel and dentine contain 051 and 0.33 per cent lipid respectively (Odutuga and Prout, 1974); these values are comparable to the values of 060 and 0.36 per cent obtained for human enamel and dentine respectively by Hess, Lee and Peckham (1956). In the present study, neutral lipids were found to represent 66 and 72 per cent of the total lipids extracted from dentine and enamel respectively. These values agree with the values of 64-66 and 72-75 per cent obtained for rat dentine and enamel respectively (Odutuga and Prout, 1973). The percentage of fatty acid in the total neutral lipids is lower than the percentage in the total phospholipids (Tables 1 and 2) owing to the inclusion of cholesterol in the total neutral lipids. In Table 2, the experimental recovery of fatty acids from the various lipids calculated as per cent of lipid is over 90 per cent in all cases when compared to the theoretical fatty acid content. The amount of fatty acid present in the teeth was calculated from the results to be 283 mg/lOO g of enamel 176 mg/lOO g of dentine. Approximately 50 per cent of total tissue lipid is fatty acid (Table 2) therefore from the above figures the lipid content of enamel should be 283 x 2 = 566 mgjlO0 g. This is similar to the figure of 571 mg/lOO g of lipid obtained from rat enamel (Odutuga and Prout, 1973) and to the figure of 530mg/lOOg of lipid extracted from human enamel (calculated from Tables 1 and 2). Similarly, the lipid content of dentine is calculated to be 352 mgjlO0 g; this also agrees with the figure of 357 mg/100 g of lipid obtained for rat dentine (Odutuga and Prout, 1973),and with the figure of 336 g/100 g of lipid extracted from human dentine (Tables 1 and 2). These figures are in the same range as the values of 510 mg/lOOg and 570 mg/lOO g of lipid extracted from human and rat enamel respectively, 330 mg/lOO g and 350 mg/lOO g of lipid extracted from human and rat dentine respectively. which have been reported previously (Prout et al., 1973; Odutuga and Prout, 1973). The difference in fatty acid composition of enamel and dentine becomes more significant when the amount of fatty acid present in the organic residue is calculated, as distinct from the total calcified tissue, since enamel contains only approximately 1.2 per cent organic matrix and dentine contains approximately 20 per cent (Jenkins, 1970). Enamel organic matrix contained approximately 23.6 g of fatty acid per 100 g, whilst dentine organic
molars
291
matrix contained 880 mg of fatty acid per 100 g. As 50 per cent of lipid in enamel and dentine is fatty acid, (23.6 x 2) = 47.2 per cent lipid is present in human enamel organic matrix and (880 x 2) 1.76 per cent lipid is present in dentine organic matrix. These figures agree closely with the percentage lipid content of 42.5 and 1.65 per cent reported previously in human enamel and dentine (Odutuga and Prout, 1973) and in rat dentine (Prout et al, 1973). The value for enamel, however, is higher than the 35.0 per cent found in rat enamel by Prout et al. (1973); this difference is not considered to be significant in view of the fact that the value for the organic matrix of rat enamel is different from that of human, rat enamel containing 1.63 per cent, and human enamel coqtaining 1.2 per cent by weight (Jenkins, 1970; Prout et al., 1973). From the results obtained in the present study, it was calculated that enamel matrix contained a concentration of fatty acid 27 times greater than that of the organic matrix of dentine. This agrees with the value of 26 times more lipid reported previously (Odutuga and Prout, 1974) and is similar to the value of 20 times more lipid reported for the rat (Prout et al., 1973). The present data provide a detailed comparison of the fatty acid composition of different lipids in the enamel and dentine of human molars. The fatty acid distribution is similar to that previously reported in total human enamel and dentine (Rabinowitz et al., 1967; Prout and Shutt, 197Oa),in total rat enamel and dentine and neutral !ipid and phospholipid of rat enamel and dentine (Prout and Shutt, 1970b; Prout and Atkin, 1973; Odutuga and Prout, 1973) and, in other hard tissues (Das and Harris, 1970a, b; Fincham et al., 1972), the major fatty acids being palmitic, stearic and oleic acid. However, these results are substantially different from the analyses of bovine bone (Shapiro, 1971) where palmitic and stearic acids were noted in smaller concentrations than were found in the present study. It has been observed in the present study that enamel contained more linoleic and linolenic acids than dentine; the greater part of these fatty acids was found in the phospholipids (mostly in the acidic phospholipids). A similar observation was made in the rat (Odutuga and Prout, 1973). In the present study trace amounts of arachidonic acid were detected in the neutral and phospholipids from human molars in contrast to the rat where the free fatty acid plus monoglycerides plus diglycerides neutral lipid fraction of incisor dentine and enamel contains 15.0 per cent arachidonic acid and the sphingomyelin plus phosphatidyl serine plus phosphatidyl inositol phospholi$d fraction of molar dentine and the phosphatidyl ethanolamine of incisor enamel contain 15.0 per cent and 7.0 per cent respectively (Odutuga and Prout, 1973). Acknowledgements-We thank Mr. P. Hancock for skilled technical assistance and the Nigerian Government for provision of a support grant for A. A. Odutuga. The work forms part of a thesis to be submitted for the degree of Ph. D. by A.A.O.
298
R. E. S. PROUT and A. A. ODUTUGA REFERENCES
Das S. K. and Harris R. S. 1970a. Fatty acids in the tooth lipids of 16 animal species. J. dent. Res. 49, 119-125. Das S. K. and Harris R. S. 1970b. Lipids and fatty acids in fossil teeth. J. dent. Res. 49, 126-130. Dirksen T. R. and Ikels K. G. 1964. Quantitative determination of some constituent lipids in human dentine. J. dent. Res. 43,246-251. Dirksen T. R. and Marinetti G. V. 1970. Lipids of bovine enamel and dentin and human bone. Calc. Zss. Res. 6, I10. Fincham A. G., Burkland G. A. and Shapiro I. M. 1972. Lipophilia of enamel matrix. A chemical investigation of the neutral lipids and lipophilic proteins of enamel. Calc. 7iss. Res. 9. 247-259. Graziano V. 1962. Determinazione gas chromatographica degli acidi grassi nella polpa di denti di bue. Boll. sot. ital. Biol. sper. 38, 13041306. Hess W. C.. Lee C. Y. and Peckham S. C. 1956. The lipid content of enamel and dentin. J. denr. Res. 35, 273-275. Jenkins G. N. 1970. The Physiology of’ the Mouth, 3rd Edi. Blackwell, Oxford. Odutuga A. A. and Prout R. E. S. 1973. Fatty acid composition of neutral lipid and phospholipids of enamel and dentine from rat incisors and molars. Archs oral Biol. 18, 689497.
Odutuga A. A. and Prout R. E. S. 1974. Lipid analysis of human enamel and dentine. Archs oral Biol. 19, in press. Prout R. E. S.. Odutuga A. A. and Tring F. C. 1973. Lipid analyses of rat enamel and dentine. Archs oral Biol. 18, 373-380. Prout R. E. S. and Atkin E. R. 1973. Effect of diet deficient in essential fatty acid on fatty acid composition of enamel and dentine of the rat. Archs oral Biol. 18, 583-590. Prout R. E. S. and Shutt E. R. 1970a. Analysis of fatty acids in human root dentine and enamel. Archs oral Biol. 15, 28 l-286. Prout R. E. S. and Shutt E. R. 1970b. Analysis of fatty acids in rat enamel and dentine. Archs oral Biol. 15, 1105-l 106. Prout R. E. S. and Shutt E. R. 197Oc. Separation of enamel and dentine using cadmium tungstoborate solution. Archs oral Biol. l&559-561. Rabinowitz J. L., Bailey T. A. and Marsh J. B. 1971. Polar and neutral lipids of pig gingiva. Archs oral Biol. 16, 1195.-1205. Rabinowitz J. L., Luddy F. E.. Barford R. A., Herb S. F., Orlean S. L. and Cohen D. W. 1967. Lipid determination in powdered human dentin by thin-layer and gas-liquid chromatography. J. dent. Res. 46, 1086-1089. Shapiro I. M. 1971. The neutral lipids of bovine bone. Archs oral Biol. 16,411421.
Rbsum&Les
lipides Heutres et les phospholipides ont ktk extraites de l’kmail et de la dentine de molaires humaines, hydrolyskes et on a dktermink la composition des acides gras des esters du cholestkrol, des triglyckides, les acides.gras libres plus les mono- et les di-glyckrides et les lipides neutres totales. On a analysk aussi la ltcithine, la sphingomyirline plus la phosphatidyle sCrine plus l’inositol phosphatidyle, Ethanolamine phosphatidyle, l’acide phosphatidique plus la cardiolipine et les acides gras phospholipidiques totaux. Les acides gras majeurs prksents ktaient les acides palmitique, stkarique et okique, palmitoltique et linokique, Ctant les constituantes majeures dans quelques phospholipides. Les proportions relatives des acides gras des lipides neutres et phospholipides ont CtC similaires dans la dentine et l’kmail. Les analyses qualitatives et quantitatives Ctaient similaires a celles obtenues des dents des rats normaux.
ZusammenfassungpEs
sind neutrale Lipide und Phospholipide aus Schmelz und Dentin von menschlichen Molaren ausgeschieden und hydrolisiert worden, und es wurden die Fettlurenzusammenstellung der Cholesterolesther, Triglyceriden, freie Fettsiiuren plus Mono- und Diglyceriden und gesamte neutrale Lipiden ermittelt. Auch sind Lezithin, Sphingomyeline plus Phosphatidylserin und Phosphatidylinosit, Phosphatidytithanolamin, Phosphatidslure plus Kardiolipine und gesamte Phospholipidefettskren analysiert worden. Die vorhandenen hauptG&lichen Fettsluren waren Palmitin, Trlnen und tjlkiuren und Palmitolein und LinolQuren waren Hauptbestandteile in einigen Phospholipiden. Die relativen Proportionen der Fetttiuren sowohl von neutralen Stoffen wie von Phospholipiden waren im Dentin und Schmelz Phnlich. Sowohl qualitative wie quantitative Analysen lhnelten denen, die von normalen Rattenzghnen erhalten wurden.