The Chemistry of Cerumen: A Preliminary Report***

THE CHEMISTRY OF CERIJMEN: A PRELIMINARY REPORT*t LEV AKOBJANOFF, C. CARRUTHERS, PHD. AND BEN H. SENTURIA, M.D.

There is some chemical and pathologic evidence to suggest that a prevalent and widely distributed form of external otitis may be caused by a malfunction of the cpidermal glands of the skin of the ear canal (1, 2). The ultimate purpose of this investigation is the development of microchemical technics for the determination of the normal constituents of ear secretions so that it may be possible to detect changes from the normal. With such information available, a means of prophylaxis and therapy may be evolved. Previous investigations of cerumen have chiefly been concerned with the lipid fraction. Nakashima (3), a pioneer in the chemical analysis of cerumen, was able to find the following constituents: ccrotic acid, cholesterol, neurostearic acid, an acid C17H3402, a substance C5H14NO2, the amino acids arginine, cystine,

histidinc, lysine, proline, tyrosine, and a "bitter substance." A recent study (4) has shown that in addition to the above amino acids, ccrumen has the following free amino acids: leucinc, isoleucine, valine, alanine, threonine, scrine, glutamic acid, aspartic acid, glycine, and y-amino butyric acid. This report deals largely with the fractionation of the lipid material of normal cerumen and with the attempts, by paper chromatography, to identify some of the fatty acids occurring in car wax. MATERIAL AND METHODS

In conformity with the most recent methods for the fractionation of the lipid material of ccrumen (5—7), the procedure shown in Diagram 1 was used in pref-

erence to extracting desiccated cerumcn, since Bloor (6) found that drying of biologic materials resulted in changes in the solubility of some of their components. Ear wax (28.5 Gm.), which had been stored at —15 to 20° C., in a deep freeze, was extracted by means of a shaking machine in a cold room (0 to 4° C.) with a 3: 1 mixture of 95 per cent alcohol and peroxide-free ethyl ether. To remove all of the lipid material the ear wax was extracted several times with the solvent mixture. The alcohol-ether soluble material was dried at room temperature over nitrogen and then re-extracted with anhydrous ether. This ether soluble fraction was redissolved in a small volume of ether and the phospholipids were separated at 0° C. by the addition of ice-cold acetone and subsequent centrifugation at 0° C. The phospholipid-free fraction was dried over nitrogen and the lipid portion thus obtained was hydrolyzed over nitrogen with alcoholic KOH by Frenche's method (8). The saponified material was then extracted with petroleum ether, in order to separate from the mixture the unsaponiflable matter *

This research was performed under Contract AF 33 (038)28643 with the USAF School of Aviation Medicine, Randolph Field, Texas. From the Department of Otolaryngology and Cancer Research, Washington University School of Medicine, St. Louis, Missouri. Received for publication March 5, 1954. 43

petroleum ether

(Water layer) Acidified with HC1 and extracted with

Sulfolipids

Cerebrosides

(Liquid)

Sphingomyelin

Lecithin Cephalia

(Solid)

Phospholipids Treated with glacial acetic acid

(Solid)

(Solid): Amino Acids

(Solid)

(Liquid) Free Amino Acids

C

Debris of skin, hair, bacteria

(Solid)

C

Reextracted with Alcohol 75 + Water 25

jtal Proteins

Main part of proteins

(Petroleum ether layer) (Water layer) Free Fatty Acids Glycerin, Cholines, etc.J ___________________ DIAGRAM 1. The proceduresused for the fractionation of fatty acids from cerumen

Higher alcohols originally esterified

onifiables

(Petroleum ether layer)

C

Hydrolysis with alcoholic KOH and extraction with petroleum ether

Total Fats

(Liquid)

Precipitated with Acetone

Lfa1 Lipids

(Liquid)

Total lipids containing soluble amino acids. After thorough drying at room temperature in nitrogen stream, reextraction with dry ether

(Liquid)

Extracted with 3:1 Alcohol Ether Mixture

C C

S C

S

uS

.cl

'TJ

C

r

z

C

CHEMISTRY OF CERUMEN

45

which consists mainly of higher alcohols, sterols, paraffins, etc. The saponifiable fraction was acidified with hydrochloric acid and re-extracted with petroleum ether to give the free fatty acids. The acidified water layer contained the water soluble substances. Paper chromatography was employed for the possible separation of the fatty acids in the saponifiable fraction obtained (following the procedure outlined in Diagram 1) since it offered the analytic advantage of detecting small amounts of fatty acids, provided conditions could be found for their separation on paper. For this purpose both pure fatty acids and the saponifiable fraction of cerumen were put on 1 X 1 cm. squares of acetone-cxtracted (Soxhiet) Whatman's No. 1 filter paper by soaking them with methanol solutions of pure fatty acids and the saponifiable fraction of cerumen. After drying, the square pieces of paper con-

taining the fatty acids were inserted into 2 slits 5 cm. from the botton end of a strip of Whatman's No. 1 paper 22" long and 1" wide. The solutions of pure fatty acids and saponifiable material of cerumen were also applied directly on the paper strip 5 cm. from the bottom of the paper. The solvent employed for the chromatography of the fatty acids consisted of 96 volumes per cent redistilled methanol and 4 volumes per cent water. This mixture was chosen primarily since Kaufmann (9), employing paper chromatography, and Holman (10), using his elution procedure, found that it produced good separation for some fatty acids. The paper strips containing the pure fatty acids or the saponifiable fraction of cerumen were hung by 12 hooks from a steel ring 6" in diameter, which was held in place by a metal stand. The hooks permitted the ends of the paper strips to

be dipped into the solvent in a petri dish. Then the stand containing the ring and hook was placed in a glass jar 15" high and 9" in diameter. Another jar of the same size was placed over this jar and a hermetic seal was made by taping a rubber strip around the junction of both jars. The solvent was then introduced into the petri dish through a hole drilled at the bottom of the top jar, as illustrated in Diagram 2. Chromatography was carried out at 40 C. in a cold room by the ascending method or by allowing the solvent to migrate up the paper strips. The solvent was allowed to ascend to within 2" of the top of the paper strips; then the jars were opened and the rack containing the paper strips removed. The paper strips were dried at room temperature. For the detection of the fatty acids on paper, both organic and inorganic sub-

stances have been employed by Kaufmann and Budwig (9, 11). The present investigators found that organic dyes such as Brom Cresol Purple, Crystal Violet, Ericheome, Cyanin R, Nile Blue Sulphate, Rhodamine B, and Victoria Blue R require delicate handling. They are sensitive to the pH of the paper and they color the background of the paper. The paper requires extensive washing and the spots produced by the action of these dyes upon the fatty acids fade quickly from the paper. In contrast to the above organic reagents, inorganic reagents, which produce insoluble soaps from the fatty acids on the paper, are much more satisfactory for

the detection of these acids. Of these inorganic reagents, lead acetate, which

46

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Iron af and

Upper jr Lower jar

Pe+ri dish

DIAGPAM OF APPARATU5 FO DL'JFLOPING PAPER CHR0MAT0GRAMS

DJAGEAM

2

TABLE

I

Characteristic reactions produced by the soaps of known fatty acids using various metals REACTING ACIDS REAGENTS

PRETREATMENT

TEMPERATTJRE

C.

U

Lead acetate + sodium suiphide.

None

Copper acetate + rubeanic acid. Ammonia vapors Iron ammonium sulfate + potassium ferricyanide

Ammonia

20°C 100°C 20°C

x x x

20°C

x

S S

0

x x x

x x x

S

20°C

S Cd

U)

x x x

x x x

x x x

x

x

x (x)

x x

x

vapors

Cobalt acetate + rubeanic acid. Ammonia vapors

0

(x)

x indicates that a characteristic spot is produced. (x) indicates a faint reaction.

proved to be the most satisfactory (Table 1), was used to determine the position or extent of migration of the fatty acids on the paper strips as follows: The paper strips were dropped into a dilute solution of lead acetate (at 200 C. for several hours, or 1000 C. for a minute or two). After washing carefully with distilled

CHEMISTRY OF CERTJMEN

47

Cerumen 28.5 Gm. Alcohol-ether extraction

Alcohol-ether soluble 20.6% (5.88 g.)

Alcohol-etber insoluble

.1

Anhydrous ether extraction

Ether insoluble Non-lipldb .i%

Ether soluble 46% (2.75 g.) Lipids, phospholipids .1.

Ice-cold acetone (to precipitate phospholipid)

Acetone soluble Total lipids 94% (2.61 g.)

Acetone insoluble 6% (0.14 g.)

Phospholipids

Saponify with alcoholic KOH and extract with petroleum ether

Petroleum ether layer

Water layer (Saponifiable fraction)

(Non-saponifiable fraction) 39% (1.02 g.)

Acidify with HC1 and extract with petroleum ether

Cholesterol (free and esterified), other sterols, squalene, waxes, high alcohols

Petroleum ether layer 61% (1.47 g.)

Free fatty acids and fatty acids from tri-

glycerides DIAGRAM 3. Relative amounts of various lipid fractions obtained from 28.5 gm. of cerumen

water, the paper strips were placed in a dilute solution of sodium sulphide, which produces permanent dark spots of lead suiphide at the locus of the fatty acids on the paper strips with practically colorless background. RE5ULTS

The relative amounts of the various fractions obtained from 28.5 Gm. of cerumen, employing the previously outlined method, are indicated in Diagram 3. It is apparent that cerumen contained only 20.6 per cent of alcohol-ether soluble material and of this fraction only 46 per cent dissolved in anhydrous ether. This ether soluble material had very little phospholipid (6 per cent). The phospholipid-free fraction contained 61 per cent saponifiable material and 39 per cent of unsaponifiable substances.

As is shown in Table II, the extraction of desiccated cerumen resulted in a wide variation in the amount of material extractable with the various solvents employed. For this reason Bloor's method was preferred to that employed by Nakashima (3). In addition to the fatty acids listed in Table I, the lead acetate proved most useful for the detection of the following fatty acids on paper: arachidic, cerotic, alpha eleostearic, erucic, lauric, lignoceric, and linolenic. As little as 0.1 mg. of these acids produced black spots on paper and as little as 0.01 mg. gave permanent, easily observable dark or brownish spots on paper.

48

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Comparison

TABLE II of the analysis of 3 samples of desiccated ear wax according to method

of Nakashima (3)

Analysis Water % Ether extract % 75% Alcohol extract

Acetone extract%

I

II

III

55.5 23.5 25.4

14.2

30.4 27.9 37.0 0.85

—

31.2 33.9

20.6

TABLE III

R values of various pure fatty acids developed at 40 C. with a mixture of methanol 96 volumes % + water 4 volumes % ACIDS

Arachidic

RF VALUES

0 0

SHAPE OE SPOT

0

Remains at origin Remains at origin Remains at origin

Lignoceric

0

Remains at origin

Stearic

0 0 0.37 0.42 0.48 0.49

Remains at origin

Behenic alpha

Eleostearic

Palmitic

Erucic Cerotic Myristic Linolenic Laurie Arachidonic Brassidic Linoleic Oleic Capric

0.49 0.50

0.51 0.53 0.58 0.60

Narrow bands Flame-like Flame-like Flame-like Oval Oval Oval Oval Oval Oval Oval

In Figure 1 are shown the relative positions of some pure fatty acids as com-

pared with a sample of cerumen fatty acids. The R, values of the pure fatty acids are listed in Table III. The term R5 is defined as the ratio of the distance traveled by a particular substance, in this case fatty acid, to the distance traveled by the solvent. From the data in Figure 1 and Table III it is apparent that arachidic, behenic, alpha eleostearic, lignoceric, stearic, and palmitic acids do not migrate on the paper under the conditions employed. Linolenic, laurie, arachidonic, brassidic, linoleic, oleic, and capric acids have RF values which vary from 0.49 to 0.60, whereas the RF values of erucic and cerotic are even smaller than those of linolenic acid. Hence the resolution of most of these fatty acids in the methanol-water mixture employed is not very good.

Nevertheless since certain pure fatty acids migrate on the paper strips to certain levels whereas other pure fatty acids remain at the origin of the paper strips, it is possible to obtain some information on the possibility of the presence of certain fatty acids in cerumen. For example, eleostearic, arachidic, behenic,

49

CEEUMRN

OF

CHEMISTRY

occur

to

known

acids

fatty

of

preparations

pure

various

acids.

fatty

wax

ear

of

in

the

human

body

in

comparison

to

those

by

produced

spots

PbS

1. FIG.

a does

as

strips

to

of

cerumen

migrates

migrate

acids

paper myristic

mixture

acid

fatty

the

of some

and

about

the

same

extent

as

the

above

acids.

It

is

therefore

possible

to

assume

that

on

the

paper

fatty strips

acids

of

cerumen,

Cerotic,

erucic,

the and

of origin

the

at

remain

acids

stearic

portion

of

and the

lignocerie,

which which

acids acids

on

the

paper

and

one

or

more

of

these

fatty fatty

pure

the

of more

or one

contain

may

cerumen

remain

at

paper.

migrate

on

the the

of origin acids

fatty

the

SUMMARY

has

been

made

on

the

nature

of

the

hpid

fraction

of

normal

cerumen.

The

alcoholprogress

some

nature,

preliminary

a

is

of

here

reported

work

the

Although

the

of

cent

per

21

some

ceru-

of

consistency

solu-

was

material

soluble

contained

solvent

latter

in

contained

portion

ether-soluble

the

Cho-

material.

the

by

identified

was

fraction

unsaponifiable

in

lesterol

reaction

and

by

infra-red

spectroseopy.

Libermann-Burchard

unsaponifiable

cent

per

39

61

per the

cent

of

saponifiable

and

Finally,

phospholipid.

of

amount

a

ble only about

in

anhydrous small

ether,

and

the

portion

soluble

the

less

than

half

of

the

alcohol-ether

only

waxy-lipid-like

rather

men.

Furthermore,

total,

a

small

amount

in

view

of

the

is

cerumen

non-desiccated

of

fraction

soluble

ether

50

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

In spite of considerable effort to identify some of the fatty acids of ear wax by paper chromatography, a good separation of pure fatty acids and also of fatty acids from the saponifiable fraction of ear wax was not obtained. However, this investigation did demonstrate that migration of certain pure fatty acids will take place on paper and that lead sulphidc is a suitable agent for the location of fatty acids on paper after migration. The authors wish to express appreciation to Dr. Sing-pao Chiang, Verna Alford and Marjorie Adler for their assistance in preparing this manuscript. REFERENCES 1. SENTTJEIA, BEN H.: Diffuse external otitis: its pathology and treatment. Tr. Am. Acad. & Otolar. 147—159, (1950).

2. SENTUETA, BEN H.: Symposium on medical treatment of otic infections. To be published. 3. NAKASHtMA, S.: TJber die chemisehe zussammensetzung des cerumens. Ztschr. f. physiol. Chem. 216: 105—109, 1933.

4. BAUEE, W. C., CAEEUTHEE5, C., AND SENTUEIA, B. H.: The free amino acid content of

cerumen. JI Invest. Dermat. 21: 2, 1953. 5. KRE5CHMEE, N.: Micro-methods in lipid chemistry. J. Am. Oil Chem. Soc. 25: 404, 1948.

6. BLOOE, W. R.: Biochemistry of fatty acids and their compounds. New York: Reinhold Publishing Company, 1947. 7. DEUEL, H. J., JE.: The lipids. Their chemistry and biochemistry, vol. I. New York: Interscience Publishers, Inc., 1951. 8. OLcOTT, H. S. AND MATTILL, H. A.: Antioxidants and the autoxidation of fats. VI. Inhibitols. J. Am. Chem. Soc. 58: 1627, 1936. 9. KAUFMANN, H. P. AND BUDwIG, J.: Die papierehromatographie auf dem fettgebiet VII. Nachweis & trennung von fettsauren. Fette u. Seifen 53: 330—339, 1951. 10. HOLMAN, R. T. AND HAGDANL, H.: Tiselius-claesson interferometrie adsorption separability of fatty acids by depressed solubility. J. Am. Chem. Soc. 72: 701—705, 1950. 11. KAUF.MANN, H. P. AND BUDwIG, J.: The foam test in paper chromatography. Fette u. Seifen 52: 555—6, 1950.

THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

94

linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.

24. Wynn, C. H. and Iqbal, M.: Isolation of rat

skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.

human epidermis, p. 511, The Epidermis

Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-

No warranty is given about the accuracy of the copy.

Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.

Cell Biol., 27: 603, 1965.

27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-

C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.

30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.

31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-

lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.

32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.

33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.

34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:

Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.

43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.

44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.

45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.

46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,

Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by

Res., 33: 176, 1964.