294 BIOCHIMICA ET BIOPHYSICA ACTA

BB* 55491

ISOLATION AND ANALYSIS OF TWO TYPES OF DIESTER WAXES

FROM THE SKIN SURFACE LIPIDS OF THE RAT*

T. NIKKARI AND E. HAAHTI

Department of Medical Chemistry, University of Tuvku, Turku (Finland)

(Received June 7th, 1968)

SUMMARY

Two types of aliphatic diester waxes have been isolated from the skin surface lipids of the rat using silicic acid chromatography. The isolated material and its al- kaline hydrolysis products were characterized using infrared spectrometry, thin-layer and gas-liquid chromatography. One type of the isolated waxes appears to be a diester of a z-hydroxy fatty acid (C,,_,,) with I molecule of unsubstituted fatty acid (C,,_,,) and I molecule of monohydric alcohol (C,,,,). The other type represents a diester of an alkane-r,a-diol (C,,_,,) with 2 molecules of fatty acid (C,,_,,). The mole- cular sizes of both types range from C,, to C,, with a maximum content at C,,_,,.

INTRODUCTION

Analysis of the lipids extracted with acetone from the hair and skin surface of the rat1 revealed the presence of a previously unknown lipid fraction, which amounted to 25-30% of the total material. In thin-layer chromatography on Silica Gel G, this fraction had a mobility intermediate to those of waxes and alkyl diglycerides, and it could be enriched from the other components of the mixture using silicic acid column chromatography. Upon alkaline hydrolysis it gave rise to both unsubstituted and hydroxy fatty acids, as well as to mono- and dihydric alcohols.

As indicated by the results, the isolated, unknown fraction-although conta- minated by other components of the mixture-was tentatively identified to be com- posed of at least 2 types of aliphatic diesters: (I) a diester of a hydroxy fatty acid with I molecule of unsubstituted fatty acid and I molecule of aliphatic monohydric alcohol, as well as (II) a diester of a dihydric alcohol with z molecules of unsubstituted fatty acid. The following structures were proposed for these compounds.

Type 1 Type II R’ R’

I I HC-O-CO-R” HC-O-CO-R”

I I 0 = C-0-CH,-R”’ H&O-CO-R”’

* A preliminary report of this work was presented at The Second Annual Meeting of Finnish Medical Societies on March joth, 1968, at Helsinki, Finland.

Biochim. Biophys. Acta, 164 (1968) 294-305

DIESTER WAXES OF RAT SEBUM 295

The present paper reports further work carried out in order to elucidate the composition and structure of these lipids. The unknown fraction of the rat skin sur- face lipids has been isolated in pure form and separated into 2 subfractions, which evidently exhibit the diester wax structures shown above.

EXPERIMENTAL

Materials All solvents and reagents were of reagent grade. The solvents were redistilled.

Reference compounds were purchased from the Applied Science Laboratories except for the following: z-hydroxy palmitic acid (Koch-Light Laboratories Ltd., purissi- mum); selachyl dioleate (Calbiochem, cat. No. 5612). Monoester waxes were syn- thesized as described earlier2. A mixture of C,_,, monohydric alcohols was prepared with LiA1Hb3 from the corresponding fatty acid mixtures (Applied Science Lab., mixtures H-104 and H-105). Tetra- and hexadecane-r,2-dials were similarly made from a-hydroxy myristic and palmitic acids, respectively.

Synthesis of diester waxes Ty$e I (I-cis-g’-docosenyl-a-octadecanoyl-hexadecanoic acid-s-01; C,,) :

W&h-%

HC-0-CO-(CH,),,-CH,

0 = C‘-0-cHe-(c~2~,-cH = cH-(cH,),,-CH,

Stearic acid (15 mg) was converted into chloride and esterified with an equi- valent amount of 2-hydroxypalmitic acid as described previously2. Unreacted 2-

hydroxypalmitic acid was removed with thin-layer chromatography. The isolated 2-octadecanoyl-hexadecanoic acid-z-01 (containing some unreacted stearic acid) was converted into chloride and esterified with an excess of cis-g-docosenol. The diester wax was recovered from a 7.5 cm x I cm Florisil column with 40 ml benzene-hexane (40: 60, by vol.) after the elution of the monoester waxes with 30 ml benzene-hexane (20:80, by vol.). The yield was 17 mg. (Colourless waxy substance, m.p. 33.5-34.5”.)

Type II (r,z-di-cis-g’-docosenoyl-hexadecane-r,z-diol; C,,) :

(S.H2)13-CH3 HC-O-CO-(CH,),-CH = CH-(CH,),,-CH,

H&O-CO-(CH,),CH = CH-(CH2)IrCH,

Erucic acid (20 mg) was converted into chloride and esterified with an equivalent amount of r,z-hexadecanediol. The synthetic diester wax was purified in a Florisil column as given for the Type I wax. The yield was IO mg. (Colourless waxy substance, m.p. 23.5-24”.)

A mixture of lauric and stearic acids was esterified with r,2-hexadecanediol to obtain a mixture of 3 diesters with Type II structure: di-dodecanoyl-r,2-diol (C,,), dodecanoyl-octadecanoyl-hexadecane-r,z-diol (C,,) and di-octadecanoyl-hexadecane- 1,2-diol (C,,).

Biochim. Biophys. Acta, 164 (1968) 294-305

296 T. NIKKARI, E. HAAHTl

Chromatogra$hic methods

Silicic acid (Mallinckrodt, IOO mesh) and Florisil (Fluka) were treated as de- scribed earlier’. Thin-layer chromatography was carried out on Silica Gel G (Merck). The spots were made visible with Rhodamine 6G or by charring with 70% bichromate- sulphuric acid. The compounds were eluted with diethyl ether from the deactivated support, which-in the case of acidic material-was acidified with acetic acid.

Gel filtration was carried out in IOO cm x 4 mm (internal diameter) glass co- lumns filled with Sephadexn LH-20 (Pharmacia, Sweden) allowed to swell overnight in chloroform containing 1% methanol. The same solvent was used for elution. The flow rate was of the order of 20 pl/min. The eluate was taken up quantitatively by a circulating chain detectort.

Gas-liquid chromatography was performed in a F and M Model 402 apparatus fitted with flame ionization detectors. The separation of the methyl esters of fatty acids, dimethyl acetals of aldehydes and trimethyl silyl ethers of alcohols was ac- complished in a 12 ft x 3 mm (int. diameter) glass column containing 3 “/b EGSS-X on siliconized Gas Chrom P (Applied Science Lab.). Gas-liquid chromatography of diester waxes was performed in a 68 cm x z mm (int. diameter) glass column filled with I y0 SE-30 on siliconized Gas Chrom P (Applied Science Lab.). The peaks were identified and quantified as described previously’. Molar percentages were calculated by assum- ing the response of the flame ionization detector to be proportional to the number of carbon atoms remaining after as many CO, groups as possible had been split offs from the derivative in question.

Determination of ester content and molecular weights

Ester-group determination was carried out according to a modification of the procedure described by SNYDER AND STEPHENS and by RENKONEN'. The samples were dissolved in toluene (0.3 ml) before the alkaline hydroxylamine reagent (I ml) was added. Hydroxylaminolysis was allowed to take place at room temperature for I h followed by the addition of ferric perchlorate reagent (2.5 ml). After 30 min at room temperature, the absorbance was read at 530 nm. Similar colour intensities were obtained from reference monoester waxes, synthetic diester waxes, methyl esters of fatty acids and triglycerides. The average absorbance corresponding to 2 pequiv. methyl arachidate was 0.588 & 0.008 (4 determinations with duplicates) when cor- rected for blank.

Number average molecular weights were calculated from the melting point depression of Exaltone (cyclopentadecanone, puriss., Fluka) in melting point capilla-

ries.

Collection of the rat skin surface ~!ipids

The skin surface lipids of living male rats weighing from IOO to 300 g were ex- tracted with acetone as described earlier’. A “blank” wash was carried out 4 days prior to each collection. After evaporation of the acetone under vacuum, the material was dissolved in hexane, dried with anhydrous Na,SO, and transferred into a tared flask. The extracts were stored in hexane at +4” under nitrogen.

Isolation of the unknown lipid fractions A total of 950 mg of the rat skin surface lipid extract was chromatographed in

Biochim. Biophys. Acta, 164 (1968) 294-305

DIESTER WAXES OF RAT SEBUM 297

Fig. r. Thin-layer chromatographic analysis of the eluate obtained during isolation of the unknown lipid fractions with silicic acid column chromatography. Unfractionated samples have been chromatographed on each side. Developing solvent: benzene-hexane (50: 50, by vol.).

Fig. 2. Thin-layer chromatography of the isolated fractions. Solvent: benzene-hexane (50: 50, by vol.). I, Isolated Fraction I; 2, a mixture of Fractions I and II eluted from the silicic acid column; 3, isolated Fraction II; 4, unfractionated rat skin surface lipid; 5, selachyl dioleate; 6, octadecyl palmitate; 7, cholesteryl palmitate.

Fig. 3. Thin-layer chromatography of the isolated fractions and synthetic diester waxes. Solvent: benzene-hexane (50:50, by vol.). I, Unfractionated rat skin surface lipid; 2, isolated Fraction I; 3, synthetic diester wax of Type I; 4, isolated Fraction II; 5, synthetic diester wax of Type Ii.

a 1.5 cm x 30 cm silicic acid column using a linear gradient from hexane to benzene- hexane (50 : 50, by vol.). Lipid fractions in the eluate were detected with the aid of a circulating chain detector-h, as well as with thin-layer chromatography (Fig. I). The contents of the fraction collector tubes containing material with thin-layer chro- matography mobility of the unknown Fractions I and II were recombined to yield {A) 237 mg lipid from tubes 28-44 (Fig. I) and (B) 37 mg lipid from tubes 45-52. (A) was subjected to rechromatography in a 1.5 cmx30 cm silicic acid column; it gave rise to 60 mg pure Fraction I (Sample I in Fig. 2) and 152 mg of a mixture of Fractions I and II (Sample 2 in Fig. 2). (B) was purified with preparative thin-layer chromatography to yield 34 mg pure Fraction II (Sample 3 in Fig. 2).

RESULTS

Thin-layer chromatography and infrared spectra of the isolated fractiom The thin-layer chromatography mobilities of the unknown fractions are inter-

mediate to those of alkyl diglycerides and monoester waxes (Fig. 2 ; Table I) and iden-

Biochim. Biojdys. Acta, 164 (1968) 294-305

298 T. NIKKARI, E. HAAHTI

TABLE I

RF VALUES OF THE ISOLATED FRACTIONS I AND II IN THIN-LAYER CHROMATOGRAPHY ON SILICA

GEL G TOGETHER WITH THOSE OF REFFERENCE COMPOUNDS

Cholesteryl palmitate Octadecyl palmitate Selachyl dioleate Batyl distearate Chimyl dipalmitate Glyceryl trioleate Glyceryl tripalmitate Synthetic diesters:

Type I Type II

Isolated Fraction I Isolated Fraction II

Benzene-l5exane

(50:50, by vol.)

0.65 1 0.56

0.07

0.34 0.25 0.34 0.24

(70:30, by vol.)

0.92

0.29

0.33 0.30 0.19 0.21

0.83 0.73

Ethyl ether-hexane

(10:90, by vol.)

0.89

0.39

0.23

0.75 0.68

tical with those of synthetic diester waxes (Fig. 3; Table I). Since dialkyl glycerides. and trialkyl glycerol were not available as reference compounds, thin-layer chro- matography in diethyl ether-hexane (IO : go, by vol.) was made in order to compare the RF values with those reported by BAUMANN et aL8 for different synthetic alkoxy- lipids. Slower mobilities were repeatedly obtained for triglycerides and alkyl digly-- cerides (RF: 0.23 and 0.39, respectively) than those given by BAUMANN et aL8 (0.31

and 0.47, respectively). Yet the RF values of the unknown Fractions I and II (0.75, and 0.68, respectively) are greater than that given by BAUMANN et aL8 for dialkyl monoglyceride (0.61) and approximately equal to that reported for trialkyl glycerol

Fig. 4. Infrared spectra of the isolated fractions. Apparatus: Perkin-Elmer Model 237. Samples: 2 06 solution in an r-mm NaCl cell or 2 mg/roo mg KBr.

Biochim. Biophys. Acta, 164 (1968) 294-305

DIESTER WAXES OF RAT SEBUM 299

(0.74). These results indicate the absence of alkyl glycerides as constituents of the isolated fractions.

In order to detect any alk-I-enyl ethers, a two-dimensional chromatogram on Silica Gel G was made according to SCHMID AND MANGOLD~; between developments the plate was exposed to HCI vapour for 20 min. No degradation products were form- ed, which indicates the absence of alk-I-enyl ethers in the fractions to be investigated. This is also shown by the infrared spectra (Fig. 4), which lack the 1640 cm-l band typical for alk-I-enyl ethers.

The infrared spectra of the unknown samples in hexane exhibit the strong C-O and C=O stretching bands characteristic of ester bonds (1158-1186 cm-l and 1750- 1755 cm-l). Additional bands due to hydrocarbon chains (716 cm-l, 1365 cm-l, 1450-1462 cm-l and z86o--2930 cm-l) are seen in the spectra from KBr pellets. There is no absorption in the 3600 cm-l region indicating the absence of free hydroxyl groups.

Analysis of the alkaline hydrolysis products Upon alkaline hydrolysis lo Fraction I (Sample 3 in Fig. 5) gives rise to mono- ,

hydric alcohols, unsubstituted fatty acids and hydroxy fatty acids; Fraction II (Sample 5 in Fig. 5) decomposes to unsubstituted fatty acids and dihydric alcohols. The hydrolysis products were isolated with preparative thin-layer chromatography. Their infrared spectra were similar to those of mixtures of reference compounds; the spectra of hydroxy fatty acids and diols are shown in Fig. 6. Gas-liquid chromatog- raphy (Figs. 7 and 8; Table II) indicated the presence of both straight and branched chains, as well as an unsaturated (mainly monoenes with straight chains) homologous series within each class of the saponification products.

Fig. 5. Thin-layer chromatography of the alkaline hydrolysis products of the isolated fractions together with reference compounds. Developing solvents: A, diethyl ether-hexane-acetic acid (90: IO: I, by vol.) ; B, diethyl ether-hexane (80: 20, by vol.) saturated with I part of 25 % NH,OH solution. For further details, see ref. 10. I, A mixture of reference (from top to bottom) aliphatic monohydric alcohols, cholesterol, fatty acids, alkane-r,2-dials and 2-hydroxy fatty acids; 2 and 6, saponification products of unfractionated rat skin surface lipid; 3, saponification products of Fraction I; 4, saponification products of a mixture of Fractions I and II eluted from the silicic acid column; 5, saponification products of Fraction II.

Biochim. Biophys. Acta, 164 (1968) 294-305

300 T. NIKKARI, E. HAAHTI

- Dihydric alcohols of fraction /I

---- A mixture Of tetro- and hexodecane-1,2-dials

30 3600 2000 lSO0 1600 1400 WA”EN”WBEA CM -’

1200 1000 800

Fig. 6. Infrared spectra of the hydroxy fatty acids and the dihydric alcohols of Fractions I and II, respectively, together with those of corresponding reference compounds. Apparatus : Perkin-Elmer Model 237. Samples: 2 yO solution in CHCl,; r-mm NaCl cell.

Unsubstituted fatty mds

Monohydric oicohols

Fig. 7. Gas-liquid chromatography patterns of the alkaline hydrolysis products of the isolated Fraction I. Column: 3 yO EGSS-X. The compounds have been chromatographed as methyl esters and/or trimethylsilyl ethers. The “carbon numbers” indicate the positions of saturated straight- chain homotogs.

Biochinz. Biophys. Acta, 164 (1968) zg&p=j

DIESTER WAXES OF RAT SERUM 301

Dihydric alcohols

Fig. 8. Gas-liquid chromatography patterns of the alkaline hydrolysis products of the isolated Fraction II. For further details, see the legend of Fig. 7.

TABLE II

MOLAR PERCENTAGE COMPOSITION OF THE ALKALINE HYDROLYSIS PRODUCTS OF THE ISOLATED

FRACTIONS I AND II*

Unsubstituted fatty acids

Fraction I Fraction II

Hydroxy fatty acids Fraction I

Monohydric alcohols Fraction I

Dihydric alcohols Fractiort II

<I4 0.2 0.6

=4 I.6 2.8 1.1 0.5 3J3 15 I.8 I.2 2.8 0.3 7.5 16 satd. 45.3 12.1 33.5 2.1 6x.9

16:1 1.6 2.1 I-T.7 3.7 17 satd. 8.7 4.7 10.7 0.1 8.7 17:1 6.3 0.4 18 satd. 21.3 x3.0 5.” 2.2 II.5 18:1-z 3.5 3.8 =?.4 I.7 19 I.1 4.7 2.3 0.8 0.6

20 satd. ‘-4 11.8 I.5 ‘4.4 0.4 20:1 0.5 I.5 0.8 21 0.7 9.2 22 satd. 1.2

::“, 0.9 18.3 o-4

22:I 0.2 2.2 0.7 23 0.5 3.6 5.0 0.1

24 satd. 3.9 9.9 24:1 0.1 4.3 2:; 25 I.3 2.3 1.6

26 satd. 3.1 I.9 26:r 0.2 2.3 ::: 27 0.4 0.4 1.0 28 satd. 0.5 0.4 1.0 28:1 0.4 I.1 5‘4 30 s&d. 0.9 30:1 5.5

32:x 3.5

* The percentages were calculated from the gas-liquid chromatography curves.

BiocMm. Biophys. Acta, 164 (1968) 294-305

302 T. NIKKARI, E. HAAHTI

The position of hydroxyl groups in the diols and in the hydroxy fatty acids was determined with periodate oxidation after the hydroxyacids had been hydrogenated and converted to diols with LiAlH,. Both gave rise to aldehydes with gas-liquid chromatography spectra very similar to those of the original mixtures, but the alde- hydes contained one carbon atom less than the original components. Since no peaks disappeared during oxidation, it was evident that the compounds under study were exclusively z-hydroxy fatty acids and 1,2-diols, respectively. This was also suggested by their infrared spectra and ther behaviour in thin-layer chromatography and gas- liquid chromatography.

Molecular weights of the isolated fractiom Molecular weight determinations using 3 independent methods (Table III) gave

quite similar number average molecular weights (846-876) for Fractions I and II. Both fractions appeared to contain 1.9-2.0 ester bonds per molecule. The reliability of the methods was shown by the determination of molecular weights of the synthetic diesters (Table IV).

TABLE III

MOLECULAR WEIGHTS OF THE ISOLATED FRACTIONS I AND II

The figures in parentheses give the number of determinations.

Fraction I Fraction II

From melting point depression : 858 f 73 (7) 846 zt 35 (7) From gas-liquid chromatography analysis* :

Hvdroxv fattv acid 279 Diol 262 Fitty adid ’ 280 Monohydric alcohol 346

Fatty acid ( :::

905 890 -2 H,O 36 36 -

869 854

From ester content** : 876 (2) 870 (2)

* The average molecular weights were calculated from the molar compositions obtained with gas- liquid chromatography (Table II). ** It was assumed that there are 2 ester bonds per molecule.

TABLE IV

MOLECULAR WEIGHTS OF THE SYNTHETIC DIESTER WAXES

The figures in Darentheses give the number of determinations.

Type I Type II

Theoretical : r-OH-palmitic acid 272 hexadecanediol- I, z 258 stearic acid 284 erucic acid 339 docosenol 315 erucic acid 339

881 936 --z H,O 36 16 2

845 9oo

From melting point depression : 832 i 49 (7) 888 & 63 (6)

From ester content: 835 (2) 909 (2)

Biochim. Biophys. Acta, 164 (1968) 294-305

DIESTER WAXES OF RAT SERUM

Fig. 4. Gasliquid chromatography patterns of the isolated fractions. The standard mixture is composed of synthetic Type II diester waxes, except the C,, compound which has the Type I structure. Stationary phase : I o/O SE-so.

Fig. 10. Gel filtration patterns of the isolated fractions and of a triglyceride mixture (glyceryl tristearate, trimyristate, tricaprate and tricaproate) . Column : Sephadex LH-so in chloroform con- taining I “/b methanol. Sample: approx. zoo ptg lipid. NGS (neopentylglycolsuccinate) was used as a void volume indicator. The compounds in the eluate were detected with the aid of a circulating chain detecto?.

Gas-liquid chromatographic analysis (Fig. g) indicates that the molecular sizes of the isolated fractions lie within a quite short range i.e. from C,, to CBS, with maximum concentrations at C,_,, (Fraction I) and C,,_,, (Fraction II). Due to the presence of both even- and odd- carbon numbered compounds, the separation was poor. The narrow molecular weight distribution is also evident from the pattern of Sephadex LH-zo chromatography (Fig. IO), according to which the (weight) average molecular weights of Fractions I and II are approx. 895 and 935, respectively, when a com- parison is made with the behaviour of a triglyceride mixture.

DISCUSSION

The most common types of neutral aliphatic diesters known to occur as con- stituents of natural lipid mixtures are the alkyl and alk-I-enyl diglyceridesll. Also diesters of ethanediol, propanediol and butanediol have been shown to be present in both plants, microorganisms and animal tissues 12. The occurrence of diesters of longer chain diols or of hydroxy fatty acids has not been conclusively demonstrated. Human vernix caseosala contains a nonpolar lipid fraction liberating fatty acids and alkane diols upon saponification and it was considered to represent a diester. NICOLAIDES~~

claims the possible presence of diesters of hydroxy fatty acids in the skin surface lipids of the rat. The latter material was studied in detail by NIXKARI~; a fraction

Biochim. Biophys. Acta, 164 (1968) 294-305

304 T. NIKKARI. E. HAAHTI

giving rise to hydroxy fatty acids and dihydric alcohols upon hydrolysis was assumed to represent a mixture of two types of aliphatic &esters.

In the present study the 2 fractions of rat sebum assumed to be composed of &ester waxes were isolated from each other and from other lipids in the mixture. Together they constituted 26% of the original mixture of rat skin surface lipids. The results of subsequent analyses of these fractions are consonant with the structures proposed. Their chromatographic behaviour is similar to that of synthetic &ester waxes. Both fractions contain r.g--2.0 ester bonds per a number average molecular weight of approx. 850470. They do not contain free hydroxyl groups, but degrade to z-hydroxy fatty acids and monohyclric alcohols (Fraction I) or r,2-dials (Fraction II) in addition to unsubstituted fatty acids, when subjected to alkaline hydrolysis.

Alkyl and alk-I-enyl glycerides have been reported to be present in the lipid mixture obtained from the preputial gland of the ratIs; this gland is a modified seba- ceous gland. These lipids, however, are obviously not present in the isolated cliester fractions. The small fraction moving next to the &ester waxesin thin-layer chromatog- raphy of the rat skin surface lipids might represent alkyl and/or alk-r-enyl diglycer- ides.

The chain length distribution of the isolated diester waxes corresponds to a number average molecular weight range from 735 to 1015, with maximum content at 820-875. This is similar to the molecular weight distribution of natural triglyceride mixtures. Since triglycerides are practically absent from rat skin surface, the &ester waxes can be considered as their “substitutes”. On human skin surface, a rapid hydrolysis of triglycerides into free fatty acids takes place, probably by a lipase of bacterial origin. The rat sebum contains only traces of free fatty acids.

In addition to the rat, r,z-diols have been isolated from the skin surface lipids of other laboratory animals as well 16. The uropygial gland of birds contains 2,3- diolsr7. Preliminary experiments have indicated the presence of lipid fractions pos- sessing the thin-layer chromatographic behaviour of &ester waxes in these materials. The Chester waxes of both types might be mistaken for alkyl cliglycericles, if hydro- genolysis with LiAlH, is performed and the alchohols liberated are studied exclusively by means of thin-layer chromatography, in which the n-monoalkyl glycerols have mobilities similar to those of alkane-r,z-diols.

ACKNOWLEDGEMENTS

We wish to thank Dr. C. G. NORDSTROM, Clinical Laboratory, Central Hospital, University of Turku, for equipment and advice in obtaining the infrared spectra.

This work was supported by a grant (HE-6818-06) from The National Institutes of Health, Bethesda, Ma., U.S.A., and by a grant from The Finnish Medical Research Council.

REFERENCES

I T. NIKKARI, Stand. J. Clin. Lab. Invest., Suppl. 85, 17 (1965). 2 E. HAAHTI, Stand. J. Clin. Lab. Invest., Suppl. 59, 13 (1961). 3 W. E.LINK,H.M.HICKMAN AND R.A.MORRISSETTE, J.Am.Oil.Chemists’Soc., 36(195g) XI. 4 E. HAAHTI, T. NIKKARI AND J. K~RKK~INEN, J.Gas Chromatog., 4 (1966) 12. 5 A. B. LITTLEWOOD, Gas Chromatography, Academic Press, New York, 1962, p. zg+ 6 F. SNYDER AND N. STEPHENS, Biochim. Biophys. Acta. 34 (1959) 244.

Biochim. Biophys. Acta, 164 (1968) 294-305

DIESTER WAXES OF RAT SEBUM 305

y 0. RENKONEN, Biochim. Biofiizys. Acta, 54 (1961) 361. 8 W.J. BAUMANN,H.H.O.SCHMID,H. W.ULSHBFERAND H.K.MANGOLD, Biochim. Biophys.

Acta, I44 (1967) 355. 9 11. H. 0. SCHMID AND W. K. MANGOLD, Biochim. Biophys. Acta, 125 (x960) 182. IO T. NIKKARI AND E. HAAHTI,J. Ch~omatog., 36 (1968) 244. II H. K. MANGOLD AND W. J. BAUMANN, in G. V. MARINETTI, Lipid Chromatographic Analysis,

Marcel Dekker, New York, 1967, p. 339. I2 I.D. BERGELSON,~. A.VAVER,N.V. PROKAZOVA, A.N. USHAKOVAND G. A.POPKOVA, Bio-

chim. Biophys. Acta, 116 (1966) 511. 13 J. K~RKK~INEN,T.NIKKARI,S.R~PONEN AND E. HAAHTI,J.IBV&. DwmafoE., 44jrg65) 333. 14 N. NICOLAIDES, J. Am. Oil Chemists Sot., 42 (rgG5) 6gr. 15 F. M. HELMY AND M. H. HACK, Comp. Biochem. Physiol., 23 (1967) 329. 16 V. R. WHEATLEY, J. Sot. Cosmetic Chemists, IO (rg5g) 206. 17 E. 0. A. HAAHTI AND H.M. F.ILES, J. Lipid lies., 8 (1967) 131.

Biochim. Biophw. Acta, 164 (1968) 294-305

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