effects of dietary restriction on the composition of lipids in rat teeth

5
EFFECTS OF DIETARY RESTRICTION ON THE COMPOSITION OF LIPIDS IN RAT TEETH S. K. DAS* and R. S. HAKKIS~ Department of Nutrition and Food Science. Massachusetts lnstltutc of Tcchnologq. Cambridge. Massachusetts 02139, U.S.A. Summary---The effect of food restriction on the quality and quantity of lipids in the teeth of ruts was studled. Control animals were fed a nutritionally adequate diet trd /ihitu,l~ while test animals were restricted to 65 per cent as much diet day by day. Both groups were started on the regimen at weaning. Approximately half were sacrificed at 43 days of age. and the remainder at 7X days of age. Compared to control group, the rats fed the restricted diet gained less body weight and retained less lipids in teeth. The tooth lipids of test animals were predominantly phospholipids and cholesterol. while those of control animals were mostly triglycerides and phospholipids. Pal- mitic, stearic and oleic acids were the major fatty acids in the teeth of rats of both groups. The ratio of oleic acid to total fatty acids in teeth was higher in the test animals than in the control animals, and it increased sharply in the teeth of control animals as they advanced from 43 to 78 days of age. However, the fatty acid composition of tooth lipids of test animals did not chnngc significantly during this period. Thus. food restriction can affect the lipid composition of the teeth of rats. The structural lipids (phospholipids and cholesterol) arc retained while the storage lipids (triglycerides) arc reduced. I\TRODUCTlOh Several studies on the lipid composition of teeth have been reported (for example. Hess, Lee and Peckham. 1956: Dirksen, Ikels and Weber, 1961; Dirksen and Ikels. 1964; Shapiro, Wuthier and Irving, 1966). Fatty acid analyses of the tooth lipids of several animal spe- cies including humans have also been conducted (Das. 1966: Rabinowitz rf ul., 1967; Das and Harris, 1970a. 197Ob; Prout and Shutt, 1970). In previous investiga- tions (Das. 1966; Alam and Harris, 1972: Prout and Atkin, 1973) it was found that diets affect the lipid and fatty acid composition of teeth. The purpose of the present investigation was to determine to what extent dietary restriction can influence the lipid composition of teeth. MATERIALS AlriD METHODS Fifteen female CR ~SD rats were placed in breeding cages when I5 days pregnant. and fed Purina Labora- tory Chow and distilled water ud [ihitum until 2 days after the pups were delivered. The pups were then dis- tributed randomly among mothers to minimize genetic influences. Litters were reduced to eight pups each (four male and four female). When the pups were 8 days old, the mothers and their litters were fed a nutri- * Present address: Department of Biochemistry and Nutrition, Mrharr! Medical Collepe. Nashville, Tennessee 37208. To whom inquiries should be directed. t Present address: Director. Nutritional Research. Mira- Im (‘ompany. Hudson. Massachusetts 01749. tionally adequate diet (Table 1) until the pups were weaned at 21 days of age. The pups were then assigned to two groups, designated “control” and “restricted”. On the first day both groups were fed (ld lihinrrn. On the second day and each day thereafter. the control group was continued on diet rlrl lihittox while each male and fcmalc of the test group was offered 55 per cent of the average amount of diet consumed by the males and females of the control group during the pre- vious day. The rats were weighed weekly between the 2lst day until the end of the experiment. Table I. Composition of experimental diet Per cent by weight Casein 20.0 Corn Starch 56.6 I Sucrose 5.0 Crisco 15.0 Salt mixture* 2.69 Vitamin mixture+ 0.5 Cystinc 0,’ ___ * Salt mixture, Bernhart and Tomarelli (1965). t Vitamin mixture (mg/lOOgm duct): B, -~-0.4; B2- 0.5; B,--0.4; B,z (Ol”+ 30; niacin -3G; Ca- pantothenate---~2.0: PABA- 10.0; inositol 200: bio- tin-~ 0.003: choline chloride---X)0.0: Menadione -0.3: folic acid- JJ35 ; DL-alpha tocopherol acctatc I OG: Dz - 125 USP units/lOOgm: and A acetate-m 1250 USP units!100 gm diet.

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Page 1: Effects of dietary restriction on the composition of lipids in rat teeth

EFFECTS OF DIETARY RESTRICTION ON THE COMPOSITION OF LIPIDS IN RAT TEETH

S. K. DAS* and R. S. HAKKIS~

Department of Nutrition and Food Science. Massachusetts lnstltutc of Tcchnologq. Cambridge. Massachusetts 02139, U.S.A.

Summary---The effect of food restriction on the quality and quantity of lipids in the teeth of ruts was studled. Control animals were fed a nutritionally adequate diet trd /ihitu,l~ while test animals were restricted to 65 per cent as much diet day by day. Both groups were started on the regimen at weaning. Approximately half were sacrificed at 43 days of age. and the remainder at 7X days of age. Compared to control group, the rats fed the restricted diet gained less body weight and retained less lipids in teeth. The tooth lipids of test animals were predominantly phospholipids and cholesterol. while those of control animals were mostly triglycerides and phospholipids. Pal- mitic, stearic and oleic acids were the major fatty acids in the teeth of rats of both groups. The ratio of oleic acid to total fatty acids in teeth was higher in the test animals than in the control animals, and it increased sharply in the teeth of control animals as they advanced from 43 to 78 days of age. However, the fatty acid composition of tooth lipids of test animals did not chnngc significantly during this period. Thus. food restriction can affect the lipid composition of the teeth of rats. The structural lipids (phospholipids and cholesterol) arc retained while the storage lipids (triglycerides) arc reduced.

I\TRODUCTlOh

Several studies on the lipid composition of teeth have been reported (for example. Hess, Lee and Peckham. 1956: Dirksen, Ikels and Weber, 1961; Dirksen and Ikels. 1964; Shapiro, Wuthier and Irving, 1966). Fatty acid analyses of the tooth lipids of several animal spe- cies including humans have also been conducted (Das. 1966: Rabinowitz rf ul., 1967; Das and Harris, 1970a. 197Ob; Prout and Shutt, 1970). In previous investiga- tions (Das. 1966; Alam and Harris, 1972: Prout and Atkin, 1973) it was found that diets affect the lipid and fatty acid composition of teeth. The purpose of the present investigation was to determine to what extent dietary restriction can influence the lipid composition of teeth.

MATERIALS AlriD METHODS

Fifteen female CR ~SD rats were placed in breeding cages when I5 days pregnant. and fed Purina Labora- tory Chow and distilled water ud [ihitum until 2 days after the pups were delivered. The pups were then dis- tributed randomly among mothers to minimize genetic influences. Litters were reduced to eight pups each (four male and four female). When the pups were 8 days old, the mothers and their litters were fed a nutri-

* Present address: Department of Biochemistry and Nutrition, Mrharr! Medical Collepe. Nashville, Tennessee 37208. To whom inquiries should be directed.

t Present address: Director. Nutritional Research. Mira- Im (‘ompany. Hudson. Massachusetts 01749.

tionally adequate diet (Table 1) until the pups were weaned at 21 days of age. The pups were then assigned to two groups, designated “control” and “restricted”. On the first day both groups were fed (ld lihinrrn. On the second day and each day thereafter. the control group was continued on diet rlrl lihittox while each male and fcmalc of the test group was offered 55 per cent of the average amount of diet consumed by the males and females of the control group during the pre- vious day. The rats were weighed weekly between the 2lst day until the end of the experiment.

Table I. Composition of experimental diet

Per cent by weight

Casein 20.0 Corn Starch 56.6 I Sucrose 5.0

Crisco 15.0 Salt mixture* 2.69 Vitamin mixture+ 0.5 Cystinc 0,’

___ * Salt mixture, Bernhart and Tomarelli (1965). t Vitamin mixture (mg/lOOgm duct): B, -~-0.4;

B2- 0.5; B,--0.4; B,z (Ol”+ 30; niacin -3G; Ca- pantothenate---~2.0: PABA- 10.0; inositol 200: bio- tin-~ 0.003: choline chloride---X)0.0: Menadione -0.3: folic acid- JJ35 ; DL-alpha tocopherol acctatc I OG: Dz - 125 USP units/lOOgm: and A acetate-m 1250 USP units!100 gm diet.

Page 2: Effects of dietary restriction on the composition of lipids in rat teeth

132 S. K. Das and R. S. Harris

Preparatiorl i$samples

Thirty-four rats from each group were sacrificed at 43 days of age. The remaining rats of each group were killed at 7X days of age. The soft tissues were removed from the jaws by a sharp blade and then the jaws were exposed to a ficin solution prepared according to the formula of Navia rt al. (1963). The four incisor and 12 molar teeth of each jaw were then removed by dissec- tion, washed with distilled water, dried in a vacuum desiccator and weighed. After weighing, the teeth were split open with a sharp scalpel and the pulp was removed by thorough scraping under a magnifying glass. Carious portions, if any, were excavated and dis- carded, The cementum was also removed by scraping with a scalpel. The depulped incisors and molars from the rats ofeach group were then pooled separately, and pulverized in a stainless-steel ball mill to pass through a 2OOmesh sieve. The teeth from males and females were pooled because the sample size was too small to permit individual analysis, and because there are no significant differences in the lipid composition of teeth according to sex at this age (Das S. K., unpublished observation).

Estructiorz of’ lipids

The tooth powders (1-2 g) were decalcified at 37°C for 24 hr with 5 vol of I5 per cent EDTA (pH 7.4); then they were refluxed for 3 hr with 5 vol of chloroform- methanol (2: 1. v/v) according to Das and Harris (I 970a). The solution was then filtered and the residue washed 3 times with 10 ml chloroform-methanol solu- tion (2: 1, v/v). The combined filtrates were evaporated to dryness and the residue thereby obtained was reex- tracted with 50 ml chloroform. The chloroform extract was washed 3 times with 10 ml distilled water and labelled as the first extract. The residue obtained after the filtration was washed 3 times with lOm1 distilled water, refluxed for 3 hr with 20 ml of 3 N H,SO, in methanol, and filtered. The filtrate was extracted 3 times with 40ml chloroform in a separatory funnel. The combined chloroform extracts were washed with water as before and labelled the second extract. The first and second chloroform extracts were combined and placed in cellulose tubing (4% nm pore diameter), and dialysed against distilled water with constant stir- ring at 4~ C for 24 hr. The solution left in the dialysis bag was transferred to a preweighed round bottom flask (250 ml capacity) and evaporated to dryness un- der nitrogen. The residue was dried in a vacuum desic- cator and weighed.

7llirl layer chrorrmtography qf‘lipids

The lipids were dissolved in a minimal volume of chloroform, and an aliquot containing approximately 200 pg of lipid was spotted on a glass plate coated with silica gel G. At both sides of the plate, various authen- tic standards were placed. The glass plate, when thor- oughly dried, was placed in a developing tank contain- ing petroleum ether-ethyl ether (80:20, v/v); the plate

was kept in this tank until the solvent reached the upper portion of the plate (1 hr). Potassium dichro- mate/sulfuric acid was used as the charring agent to visualize the lipids.

The lipids were saponified, the released fatty acids were esterified with 2 N anhydrous HCl in methanol. and analyzed by gas-liquid chromatography. The column consisted of 15 per cent diethylene glycol suc- cinate on 8(t 100 mesh gas Chrom P. A Barber-Col- man Model IO gas chromatograph equipped with an argon ionization detector was used as reported by Das ( 1966) and Das and Harris ( 197Oa).

RESULTS AYD DISCUSSION

The data relating to the effect of dietary restriction on the body weight and tooth weight gain of rats are shown in Table 2. At the end of period I (43 days of age), the males and females of the restricted group showed respectively 43 and 42 per cent less gain in body weight than the control animals. whereas in period 2 (78 days of age). the males and females of the restricted group had approximately 67 and 54 per cent less body weight gain than the control animals. No sig- nificant etrect of dietary restriction was observed on the molar tooth size of male and female rats, and on the incisor tooth size of the female rats in either of the two experimental periods. despite large caloric restric- tion. The male rats of the restricted group had only 8 and 20 per cent less incisor tooth weight gain in the first and second periods respectively than the corre- sponding controls. This observation was not surprising since the molar teeth had been mainly formed before the experiment began. However, it is of interest for the rapidly-growing incisors. since they were completely replaced during the 57 day experiment.

Data of the lipid and fatty acid content of the teeth of the rats killed at 43 and 78 days of age are shown in Table 3. It is evident that the lipid and fatty acid content of both incisors and molars were higher in the control group than in the restricted group. Also, it will be noted that though the amounts of lipids and fatty acids of the control teeth were higher at 78 days of age than at 43 days of age, no such change was noticed in the restricted group. In the rats killed 22 days after the onset of the experiment, portions of the incisors and third molar crowns of the test rats were formed during the period of caloric restriction. and could have been so affected. For the molar teeth, any reduction in lipids can be mostly explained on the basis of with- drawal or catabolism in tissues already formed. In the incisors, a reasonable explanation is that less lipids and fatty acids were incorporated during formation. Thin layer chromatographic studies of lipids revealed that (1) the incisors and molars of the control group con- tained a greater variety of lipids (cholesterol esters. triglycerides, free fatty acids. cholesterol, di- and monoglycerides and phospholipids) than those of the

Page 3: Effects of dietary restriction on the composition of lipids in rat teeth

Effects of dietary restriction in rat teeth

Page 4: Effects of dietary restriction on the composition of lipids in rat teeth

S. K. Das and R. S. Harris

Page 5: Effects of dietary restriction on the composition of lipids in rat teeth

Effects of dietary restriction m rat teeth I.15

restricted group (where the major identifiable lipids were cholesterol and phospholipids), (2) the major lipids in the teeth of control animals were triglycerides and phospholipids and thus (3) phospholipids and cho- lesterol are the chief types of functional or structural lipids in teeth.

Data of the effect of dietary restriction on the fatty

acid composition of tooth lipids in rats are shown in Table 4. The fatty acid composition of even the control rats was considerably different from that reported by Das and Harris (197Oa), particularly in regard to C 16:O and C16: 1. These differences are likely due to diffcrenccs in nutritional variables, such as age and diet between the two studies. In the present study, the inci- sors and molars of animals of both groups contained at lcast trace amounts of all of the fatty acids between 12 and 26 carbon atoms long which are commonly found in animal tissues, and they contained zero to five unsaturated bonds;‘molecule. The major fatty acids in the teeth were palmitic. stearic and oleic acids. There were no remarkable differences in the fatty acid com- position between incisors and molars in either group. The proportion of oleic acid of total fatty acids in teeth wah higher in test animals than in control animals. The proportion of oleic acid of total fatty acids increased sharply in teeth of control animals during 43-78 days of age. But the fatty acid distribution of total lipids did not change significantly in the teeth of test animals during that growth period.

It appears that food restriction can affect the lipid and fatty acid content of the teeth ofrats. The retention of phospholipids and cholesterol, and the loss of trigly- cerides during dietary restriction, suggest that phos- pholipids and cholesterol are structural lipids in teeth whereas triglycerides are a source of energy, and hence arc the storage lipids. These observations, together with others, such as (I) phospholipids are present in higher amounts in the enamel matrix of teeth than in dentin (Shapiro (‘I trl.. 1966). (2) enamel lipids are less saturated than dentin lipids (Leopold, Hess and C‘arter. I Y 5 I ) and (3) phospholipids are the most unsa- turated lipids in teeth (Das and Harris, 1970a), suggest

that dietary restriction might have a more pronounced influence on dentin lipids than on enamel lipids.

Ackno~lrdqe~nt -This investigation was supporlrd h! grants from the National Institute of Dental Research (DF- 01438).

REFERENCES

Alam S. Q. and Harris R. S. 1971. Effects of nutrition on the composition of tooth lipids and fatty acids in rats I: The effects of dietary carbohydrates on fdtt) acid colnposl- tion of rat teeth. J. dent. Res. 51, 1474 1477.

Bernhart F. A. and Tomarelli R. M. 1963. A salt mlxturc with a composition calculated from cstimatc!. 01 the mineral requirement of the rat. Ferl. Proc,. 24, 373.

Das S. K. 1966. Factors affecting the fatty acid compohitmn of teeth. Sc.D. Thesis. M.I.T.. Cambridge. Mass.

Das S. K. and Harris R. S. 1970~ Fatty acids in tooth liplds of I6 animal species. J. drnt. Rrs. 49. 119~ 125.

Das S. K. and Harris R. S. 1970b. Lipids and fatty actda in fossil teeth. J. dent Rrs. 49, l&130.

Dirksen T. R.. lkels K. G. and Weber T. B. 1961. The ~sola- tion and identification of oral lipid components. .I ~/VIII. Rex 40, 694.

Dlrksen T. R. and Ikels K. G. 1964. Quantltativc detcrmnx- tion of some constituent lipids in human dentin. ./. t/c~t. Rex 43, 346151.

Hess W. C.. Lee C. Y. and Peckham S. c‘. IC)%. The lipld content of enamel and dentin. J. rlrnf. t&. 35, 273 275.

Leopold R. S., Hess W. C. and Carter W. J. 1951. Dentinal protein: bound cholesterol. J. dent. Rcs. X0, X37 X39.

Navia J. M.. Harris R. S., Nizel A. E. and Moor J. R. 1963. Technique for removal of intact teeth from jaws of cxper- imental animals. J. drnt. Res. 42, 12.51.

Prout R. E. S. and Shutt E. R. 1970. Analysis of fatt! acids in rat enamel and dentin. ilrchs orcil Biol. 15, I IO5 I I Oh.

Prout R. E. S. and Atkin E. R. 1973. Eflect of diet deticlcnt in essential l’dtty acid on fatty acid composition of cnamcl and dentine of the rat. Archs ortrl Biol. 18, 583 590.

Rabmowitr J. L. Luddy R. E.. Barford R. A.. Herb S. F.. Orlean S. L. and Cohen D. W. 1467. Lipid detcrmlnatlon in powdered human dentin by thin-laqcr and la\-liquid chromatography. J. dvr~r. Res. 46, 1086 1089.

Shapiro I. M.. Wuthier R. E. and Irvmg J. T. I. 1966. I’namcl matrix and dentin. 4rc/1.\ orctl Bid. II. 501 512.