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Lipids of the uropygial gland of birds
Comp. Biochem. Physiol.,
1964, Vol. 12, pp. 435 to 437. Pergamon Press Ltd. Printed in Great Britain
L I P I D S OF T H E U R O P Y G I A L G L A N D OF BIRD S E. HAAHTI,* K. LAGERSPETZ,* T. NIKKARI* and H. M. FALES Department of Medical Chemistry and Department of Zoology, University of Turku, Finland, and Section of Chemistry, National Heart Institute, Bethesda, Md., U.S.A. (Received 19 February
1964)
Abstract--1. Uropygial gland lipids of fourteen species of birds were analyzed with thin layer chromatography. In most of the analyzed species the main lipid fractions were aliphatic waxes. 2. In three of the analyzed species belonging to Galliformes another type of "waxes" was present. These waxes were shown to consist of esters of dihydric alcohols with fatty acids. 3. Removal of the uropygial gland from chicks was observed to cause a statistically significant increase in growth when commercial chick-food was fed a d libitum to the operated and sham-operated chickens.
INTRODUCTION THE uropygial gland (preen gland) is the sole sebaceal gland of birds. The lipids of the gland as well as the biochemistry and physiology of this gland are not particularly well known. The chief interest has been directed to studies on the preen gland functions of waterfowl, especially the duck. Nowak (1962) has reviewed the data on the chemistry of duck preen gland lipids. Histochemical studies have been carried out by B6 (1953) on the uropygial gland of P h a l a c r o c o r a x b r a s i l i a n u s and by Kanwer (1961) on that of pigeons and domestic fowl. So far no characterization of the native preen gland lipids with newer analytical techniques has been attempted. Also the physiological significance of the preen gland secretion is still unsettled. Current theories (feather waterproofing, ergosterol production, sexual attraction, assistance in ecdysis) date back to Hou (1928). The extirpation of the preen gland of duck results not only in defatting and subsequent hydrophilia of the feathers but also in a metabolic disorder characterized by Hou (1929) as rickets. In mammals the lipid composition of the sebaceal gland secretion is shown to be specific for different species and races (Wheatley, 1956). The present work is based on our earlier studies on the chemistry of skin surface lipids (Haahti, 1961 ; Haahti &Horning, 1963). A comparative scanning of the preen gland lipids of some birds representing different families as well as the effect of the extirpation of the preen gland of chickens was thus chosen as the subject of this study. * Research Fellow of the National Science Council of Finland. 29
435
436
E. HAAHTI,K. LAGERSPETZ,T. NIKKARIANDH. M. FALES
MATERIALS AND METHODS Lipids were extracted in chloroform-methanol (2 : 1) from the contents of freshly prepared uropygial glands of the following fourteen species representing eight orders of birds : Colymbiformes Red-throated diver (Gavia stellata) Podicipitiformes Horned grebe (Podiceps auritus) Anseriformes Domestic goose (Anser anser) Domestic duck (Anasplatyrhyncos) Goldeneye (Bucephala clangula) Falconiformes Kestrel (Falco tinnunculus) Sparrow hawk (Accipiter nisus)
Galliformes Domestic hen (Gallus domesticus) Partridge (Perdix perdix) Pheasant (Phasianus colchicus) Lariformes Lesser black-backed gull (Larus fuscus) Strigiformes Tengmalm's owl (Aegolius funereus) Long-eared owl (Asio otus) Passeriformes Jackdaw (Coleus monedula)
Purification of the lipids was carried out according to the method of Folch et al. (1957). The lipids were then chromatographed on silicic acid and the neutral lipid fraction was eluted with chloroform (Haahti, 1961) for further analysis with thin layer chromatography on Silica gel G (Merck). Details of the conditions of thin layer chromatography are given in the legends of the figures. The uropygial gland was extirpated from twelve chicks aged 2 weeks, using ethyl-ether anaesthesia. Eleven similar controls were sham-operated simultaneously. Each group included five cockerels. The animals were weighed at intervals up to the age of 2 months. Water and commercial chick food were available ad libitum in the animal cages. RESULTS AND DISCUSSION Figure 1 shows the fractionation of the uropygial gland lipids of the fourteen bird species with thin layer chromatography. In all samples except the Galliformes (samples 8, 9, 10) the chief neutral lipid fraction consists of aliphatic waxes. The hydrolysates of these waxes were shown to consist mostly of aliphatic alcohols and fatty acids as analyzed with thin layer chromatography. In samples Nos. 4 and 11 (Domestic duck, Lesser black-backed gull) the nonpolar wax moiety fell in two fractions. This may be caused by the presence of esterified hydroxy fatty acids, as described in other reports by us (Nikkari & Haahti, 1964). The main lipid component of samples 8, 9 and 10 (Galliformes) was a fraction which in thin layer chromatography is located between the waxes and the triglycerides. With a better resolution this fraction divides in two spots of about eqtial magnitude. A sample of chicken uropygial gland lipid (Fig. 2) was subjected to silicic acid column chromatography (Haahti, 1961) in order to purify the above double-fraction. After a rinse with 10 column volumes of 2% (v/v) benzenehexane the fraction was recovered in an eluate of 30% (v/v) benzene-hexane. An alkaline hydrolysate of the obtained material showed fatty acids ranging from Ca0 to C~0 and a nonsaponifiable fraction. The nonsaponifiable fraction has in thin layer chromatography a very similar mobility as reference 12-OH hydroxylalcohol.
SOLVENT
-FATTY
FRONT
AC10
METHYL
ESTERS
TRIGLYCERIDES ALPHATIC ALCOHOLS CHOLESTEROL
FIG. 1. Thin layer chromatogram of preen gland lipids of the following birds: (1) Red-throated diver (Gacia stelluta), (2) Horned grebe (Pod&s auritus), (3) Domestic goose (Anser anser), (4) Domestic duck (Anaspluryrhyncos), (5) Goldeneye (Bucephulu clungzclu), (6) Kestrel (F&o tinnuncz~lus), (7) Sparrow hawk (Accipiter nisus), (8) Domestic hen (Gulbls domesticzcs), (9) Partridge (Per& per&), (10) Pheasant (Phusiunzcs colchiczcs), (11) Lesser black-backed gull (Luvus f~scus), (12) Tengmalm’s owl (Aegolirts fitnereus), (13) Long-eared owl (Asio otus), (14) Jackdaw (Coleus monedzdu). Conditions of analysis: Support: 0.25 mm layer of Kiesel gel G (Merck). Run in S-chamber (Desaga). Samples about 100 pg each. Staining: Sulphuric acid-bichromate.
SOLVENT
FRONT
FIG. 2. Thin layer chromatography of (1) major nonsaponifiable constituent of chicken uropygial gland lipid (see text), (2) batyl-alcohol and (3) oleyl alcohol. Solvent: Ethyl acetate-chloroform 1 : 4. Other conditions as in Fig. 1.
437
LIPIDS OF THE UROPYGIALGLANDOF BIRDS
In gas-chromatography (6 ft column of 1 per cent SE 30 at 225°C) the nonsaponifiable fraction shows four homologue peaks in the range of C,,-C,,. Acetylation of the material results in increase of retention times, and this increase corresponds to the introduction of two acetyl-groups on each of the four fractions. The extirpation of the uropygial gland resulted in no unusual appearance or habits of the chickens, nor were any symptoms or signs of rickets detectable comThe mean weight of the operated chick pared with the sham-operated group. at the age of 2 months was 1017 g + 11.6 g while that of the controls was only 893.6 _+27.9 g. The difference between the means was statistically significant (t = 4.11, p< 0.001). It seems to us possible that this somewhat unexpected effect can be caused by an increased need of dietary antirachitic factors, resulting in an increased food consumption and faster growth. Acknowledgements-This investigation was supported by PHS Research HE-06818-02 from The National Heart Institute, Bethesda, Md., U.S.A.
Grant
No.
REFERENCES B6 N. A. (1953) Observaciones sobre la glandula uropigia de1 vigua Phalucrocorux brusiZiunus brusiliunus (Gmelin). Cienc. e Invest. 9, 521-524. FOLCH J., LEES M. & STANLEY G. H. S. (1957) A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509. HAAHTI E. (1961) Major lipid constituents of human skin surface. &and. J. Clin. Lab. Invest. 13, Suppl. 59. HAAHTI E. & HORNING E. C. (1963) Isolation and characterization of saturated and unsaturated fatty acids and alcohols of human skin surface lipids. &and. J. C&z. Lab. Invest. 15, 73-78. Hou H. C. (1928) Studies on the glandula uropygialis of birds. Chin.J. Physiol. 2, 345-380. Hou H. C. (1929) Relation of the preen gland (glandula uropygialis) of birds to rickets. Chin.J.
Physiol.
3, 171-182.
KANWER K. C. (1961) Morphological and histochemical studies on the uropygial glands of pigeon and domestic fowl. Cytologiu 26, 124-136. NIKKARI T. & HAAHTI E. (1964) The composition of rat skin surface lipids. Actu Chem. Scund. (In press.) NOWAK G. A. (1962) Verzweigte Fettsaure-Ester nach dem Prinzip des Biirzeldrtisenoeles von Wasservogeln. Drugoco Berichte 279-287. WHEATLEYV. R. (1956) Sebum: its chemistry and biochemistry. Amer. Perfum. 68, 37-47.
1964, Vol. 12, pp. 435 to 437. Pergamon Press Ltd. Printed in Great Britain
L I P I D S OF T H E U R O P Y G I A L G L A N D OF BIRD S E. HAAHTI,* K. LAGERSPETZ,* T. NIKKARI* and H. M. FALES Department of Medical Chemistry and Department of Zoology, University of Turku, Finland, and Section of Chemistry, National Heart Institute, Bethesda, Md., U.S.A. (Received 19 February
1964)
Abstract--1. Uropygial gland lipids of fourteen species of birds were analyzed with thin layer chromatography. In most of the analyzed species the main lipid fractions were aliphatic waxes. 2. In three of the analyzed species belonging to Galliformes another type of "waxes" was present. These waxes were shown to consist of esters of dihydric alcohols with fatty acids. 3. Removal of the uropygial gland from chicks was observed to cause a statistically significant increase in growth when commercial chick-food was fed a d libitum to the operated and sham-operated chickens.
INTRODUCTION THE uropygial gland (preen gland) is the sole sebaceal gland of birds. The lipids of the gland as well as the biochemistry and physiology of this gland are not particularly well known. The chief interest has been directed to studies on the preen gland functions of waterfowl, especially the duck. Nowak (1962) has reviewed the data on the chemistry of duck preen gland lipids. Histochemical studies have been carried out by B6 (1953) on the uropygial gland of P h a l a c r o c o r a x b r a s i l i a n u s and by Kanwer (1961) on that of pigeons and domestic fowl. So far no characterization of the native preen gland lipids with newer analytical techniques has been attempted. Also the physiological significance of the preen gland secretion is still unsettled. Current theories (feather waterproofing, ergosterol production, sexual attraction, assistance in ecdysis) date back to Hou (1928). The extirpation of the preen gland of duck results not only in defatting and subsequent hydrophilia of the feathers but also in a metabolic disorder characterized by Hou (1929) as rickets. In mammals the lipid composition of the sebaceal gland secretion is shown to be specific for different species and races (Wheatley, 1956). The present work is based on our earlier studies on the chemistry of skin surface lipids (Haahti, 1961 ; Haahti &Horning, 1963). A comparative scanning of the preen gland lipids of some birds representing different families as well as the effect of the extirpation of the preen gland of chickens was thus chosen as the subject of this study. * Research Fellow of the National Science Council of Finland. 29
435
436
E. HAAHTI,K. LAGERSPETZ,T. NIKKARIANDH. M. FALES
MATERIALS AND METHODS Lipids were extracted in chloroform-methanol (2 : 1) from the contents of freshly prepared uropygial glands of the following fourteen species representing eight orders of birds : Colymbiformes Red-throated diver (Gavia stellata) Podicipitiformes Horned grebe (Podiceps auritus) Anseriformes Domestic goose (Anser anser) Domestic duck (Anasplatyrhyncos) Goldeneye (Bucephala clangula) Falconiformes Kestrel (Falco tinnunculus) Sparrow hawk (Accipiter nisus)
Galliformes Domestic hen (Gallus domesticus) Partridge (Perdix perdix) Pheasant (Phasianus colchicus) Lariformes Lesser black-backed gull (Larus fuscus) Strigiformes Tengmalm's owl (Aegolius funereus) Long-eared owl (Asio otus) Passeriformes Jackdaw (Coleus monedula)
Purification of the lipids was carried out according to the method of Folch et al. (1957). The lipids were then chromatographed on silicic acid and the neutral lipid fraction was eluted with chloroform (Haahti, 1961) for further analysis with thin layer chromatography on Silica gel G (Merck). Details of the conditions of thin layer chromatography are given in the legends of the figures. The uropygial gland was extirpated from twelve chicks aged 2 weeks, using ethyl-ether anaesthesia. Eleven similar controls were sham-operated simultaneously. Each group included five cockerels. The animals were weighed at intervals up to the age of 2 months. Water and commercial chick food were available ad libitum in the animal cages. RESULTS AND DISCUSSION Figure 1 shows the fractionation of the uropygial gland lipids of the fourteen bird species with thin layer chromatography. In all samples except the Galliformes (samples 8, 9, 10) the chief neutral lipid fraction consists of aliphatic waxes. The hydrolysates of these waxes were shown to consist mostly of aliphatic alcohols and fatty acids as analyzed with thin layer chromatography. In samples Nos. 4 and 11 (Domestic duck, Lesser black-backed gull) the nonpolar wax moiety fell in two fractions. This may be caused by the presence of esterified hydroxy fatty acids, as described in other reports by us (Nikkari & Haahti, 1964). The main lipid component of samples 8, 9 and 10 (Galliformes) was a fraction which in thin layer chromatography is located between the waxes and the triglycerides. With a better resolution this fraction divides in two spots of about eqtial magnitude. A sample of chicken uropygial gland lipid (Fig. 2) was subjected to silicic acid column chromatography (Haahti, 1961) in order to purify the above double-fraction. After a rinse with 10 column volumes of 2% (v/v) benzenehexane the fraction was recovered in an eluate of 30% (v/v) benzene-hexane. An alkaline hydrolysate of the obtained material showed fatty acids ranging from Ca0 to C~0 and a nonsaponifiable fraction. The nonsaponifiable fraction has in thin layer chromatography a very similar mobility as reference 12-OH hydroxylalcohol.
SOLVENT
-FATTY
FRONT
AC10
METHYL
ESTERS
TRIGLYCERIDES ALPHATIC ALCOHOLS CHOLESTEROL
FIG. 1. Thin layer chromatogram of preen gland lipids of the following birds: (1) Red-throated diver (Gacia stelluta), (2) Horned grebe (Pod&s auritus), (3) Domestic goose (Anser anser), (4) Domestic duck (Anaspluryrhyncos), (5) Goldeneye (Bucephulu clungzclu), (6) Kestrel (F&o tinnuncz~lus), (7) Sparrow hawk (Accipiter nisus), (8) Domestic hen (Gulbls domesticzcs), (9) Partridge (Per& per&), (10) Pheasant (Phusiunzcs colchiczcs), (11) Lesser black-backed gull (Luvus f~scus), (12) Tengmalm’s owl (Aegolirts fitnereus), (13) Long-eared owl (Asio otus), (14) Jackdaw (Coleus monedzdu). Conditions of analysis: Support: 0.25 mm layer of Kiesel gel G (Merck). Run in S-chamber (Desaga). Samples about 100 pg each. Staining: Sulphuric acid-bichromate.
SOLVENT
FRONT
FIG. 2. Thin layer chromatography of (1) major nonsaponifiable constituent of chicken uropygial gland lipid (see text), (2) batyl-alcohol and (3) oleyl alcohol. Solvent: Ethyl acetate-chloroform 1 : 4. Other conditions as in Fig. 1.
437
LIPIDS OF THE UROPYGIALGLANDOF BIRDS
In gas-chromatography (6 ft column of 1 per cent SE 30 at 225°C) the nonsaponifiable fraction shows four homologue peaks in the range of C,,-C,,. Acetylation of the material results in increase of retention times, and this increase corresponds to the introduction of two acetyl-groups on each of the four fractions. The extirpation of the uropygial gland resulted in no unusual appearance or habits of the chickens, nor were any symptoms or signs of rickets detectable comThe mean weight of the operated chick pared with the sham-operated group. at the age of 2 months was 1017 g + 11.6 g while that of the controls was only 893.6 _+27.9 g. The difference between the means was statistically significant (t = 4.11, p< 0.001). It seems to us possible that this somewhat unexpected effect can be caused by an increased need of dietary antirachitic factors, resulting in an increased food consumption and faster growth. Acknowledgements-This investigation was supported by PHS Research HE-06818-02 from The National Heart Institute, Bethesda, Md., U.S.A.
Grant
No.
REFERENCES B6 N. A. (1953) Observaciones sobre la glandula uropigia de1 vigua Phalucrocorux brusiZiunus brusiliunus (Gmelin). Cienc. e Invest. 9, 521-524. FOLCH J., LEES M. & STANLEY G. H. S. (1957) A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226, 497-509. HAAHTI E. (1961) Major lipid constituents of human skin surface. &and. J. Clin. Lab. Invest. 13, Suppl. 59. HAAHTI E. & HORNING E. C. (1963) Isolation and characterization of saturated and unsaturated fatty acids and alcohols of human skin surface lipids. &and. J. C&z. Lab. Invest. 15, 73-78. Hou H. C. (1928) Studies on the glandula uropygialis of birds. Chin.J. Physiol. 2, 345-380. Hou H. C. (1929) Relation of the preen gland (glandula uropygialis) of birds to rickets. Chin.J.
Physiol.
3, 171-182.
KANWER K. C. (1961) Morphological and histochemical studies on the uropygial glands of pigeon and domestic fowl. Cytologiu 26, 124-136. NIKKARI T. & HAAHTI E. (1964) The composition of rat skin surface lipids. Actu Chem. Scund. (In press.) NOWAK G. A. (1962) Verzweigte Fettsaure-Ester nach dem Prinzip des Biirzeldrtisenoeles von Wasservogeln. Drugoco Berichte 279-287. WHEATLEYV. R. (1956) Sebum: its chemistry and biochemistry. Amer. Perfum. 68, 37-47.