Vol. 67

Comparative Studies of ' Bile Salts '10. BILE SALTS OF THE KING PENGUIN, APTENODITES PATAGONICA*

By I. G. ANDERSON AND G. A. D. HASLEWOODGuy's Hospital Medical School, London, S.E. 1

AND I. D. P. WOOTTONPo8tgraduate Medical School, London, W. 12

(Received 11 April 1957)

The scanty information about the chemical natureof the bile acids of birds has not been previouslycollected together and this is now done in Table 1.Claims have been made for the detection of free bileacids in bird bile (e.g. Maeda, 1938), but it is believedthat these acids generally occur conjugated withtaurine. Haslewood & Sjovall (1954) could detectno glycine conjugates in the bile salts of eightspecies of birds, and Wiggins (1955a), in a carefulexamination of chicken bile by countercurrentdistribution methods, failed to find either free bileacids or glycine conjugates.The present report is of an examination of the

bile salts of the King penguin, Aptenodites pata-gonica, and includes remarks on the chemicalnature of tetrahydroxynorsterocholanic acid.

RESULTS

By methods previously used in these studies, Kingpenguin bilewas shown to contain chenodeoxycholicacid, cholic acid and tetrahydroxynorsterocholanic

* Part 9: Haslewood (1956).

acid. At least four other 'bile acids' may be presentin the bile; none of these appeared to be substancesknown to us. The proportion of cholic and tetra-hydroxynorsterocholanic acids, taken together, wasmuch greater than in chicken bile, but their strikingchemical similarity prevented complete separationof these acids or their esters and hence any reliableestimation of the amounts of each.

Ethyl cholate and 'ethyl tetrahydroxynorstero.cholanate' ran at virtually the same rate on our

paper chromatograms. These esters apparentlyformed mixed crystals; the melting point of one ofour preparations could be raised to about 2000 byrecrystallization from benzene. Material of thispurity on hydrolysis yielded a sample of tetra-hydroxynorsterocholanic acid, m.p. about 2140,with characteristic behaviour, apparently sub-stantially the same substance as a sample earliergiven to us by Professor T. Kazuno (see Discussion).The ethyl esters of our material and that of Kazunolikewise appeared to be identical. Elementaryanalyses of our product gave figures correspondingto C26H4405, i.e. to ethyl cholate, rather than to the

SpeciesDomestic chicken

Domestic duck (Anas domestica);wigeon, Anas (Mareca) penelope;turkey (Meleagri8 gallopavo)Domestic goose

Pheasant, Phasianus colchicuskarpowiOwlKing penguin, Aptenoditespatagonica

Table 1. Bile acids of birds

Bile acids isolatedChenodeoxycholic (3a::7ax-dihydroxy-cholanic)

Isolithocholic

Cholic (3x:7a:12x-trihydroxycholanic)Tetrahydroxynorsterocholanic3oc-Hydroxy-7-oxocholanic3-Oxochola-4:6-dienic*Cholic; chenodeoxycholic

Chenodeoxycholic

Chenodeoxycholic

Cholic; chenodeoxycholicCholic; chenodeoxycholic; tetra-hydroxynorsterocholanic

ReferenceYonemura (1926); Maeda (1938);Takahashi (1938)

Hosizima, Takata, Uraki & Sibuya(1930)

Yamasaki (1933)Yamasaki (1951)Wiggins (1955a)Wiggins (1955b)Ishihara & Mori (1938)

Marsson (1849a, b); Windaus,Bohne & Schwarzkopf (1924)

Ohta (1939a)

Ono & Tamura (1953)Present work

* May be an artifact derived from 7ox-hydroxy-3-oxochol-4-enic acid (conjugated).

21-2

323

I. G. ANDERSON, G. A. D. HASLEWOOD AND I. D. P. WOOTTON

formula C29H5006 required for the ethyl ester of a'tetrahydroxynorsterocholanic acid', C27H4606.The possible significance of these analytical figuresis discussed below. An attempt was made to effectfurther purification of our 'ethyl tetrahydroxy-norsterocholanate' by partition chromatography onCelite (kieselguhr) columns, as described byCarpenter & Hess (1956); this was unsuccessful.

Oxidation by chromic acid ofa less pure sample of'ethyl tetrahydroxynorsterocholanate', of m.p.about 180-196', gave a neutral mixture meltingover a wide range. This material was partiallyseparated on Celite columns, giving apure sample ofethyl dehydrocholate (ethyl 3:7:12-trioxocholanate)and a substance of m.p. 157-166°, together with amixture. A study of the result of this separationsuggested that the ester, m.p. 180-196o, mightconsist of a binary mixture of about 57 % of ethylcholate and 43% of ethyl tetrahydroxynorstero-cholanate: however, the presence of other con-stituents could not be excluded. Further purifica-tion of 'ethyl tetrahydroxynorsterocholanate' wasnot feasible with the amounts of material available.

Infrared spectra ofthe chromic oxidation product,m.p. 157-166°, mentioned above and of pure ethyldehydrocholate were compared. The spectra showeda general similarity, but also well-marked differ-ences. A comparison between the infrared spectraof methyl cholate and of a methylated sample ofKazuno's acid has already been reported (Hasle-wood & Wootton, 1956).

EXPERIMENTAL

General. Melting points were determined on a Kofler-block type of apparatus, and are corrected. Infraredspectroscopy was carried out as described by Wootton &Wiggins (1953). A1203 (S) was 'Type H' (Peter Spence andSons Ltd., Widnes, Lancs.): A1203 (N) was also from PeterSpence and Sons Ltd., neutralized as described by Shoppee(1949). Light petroleum had b.p. 40-60°, unless otherwisestated. Chromic acid (20%) was a solution of about 20 g. ofCrO3 in a minimal volume of water made, with mixing, to100 ml. with acetic acid. Microanalyses (C, H) were byDrs Weiler and Strauss, Oxford. Bile salts were isolated asdescribed by Haslewood & Wootton (1950). 'Amyl acetate'was a commercial sample ofmixed isomers. Itwas redistilledand then had b.p. 130-140°.

Paper chromatography of bile salts. With King penguinbile salts, Haslewood & Sjovall (1954) found spots corre-sponding to taurocholate and taurodeoxycholate in asystem not distinguishing between taurodeoxycholate andtaurochenodeoxycholate. In the present work, when themethod of Sjovall (1955) for separating these substances[descending paper chromatography: mobile phase, 80%(v/v) amyl acetate-heptane; stationary phase, 70% (v/v)acetic acid-water] was used, spots corresponding to tauro-cholate and taurochenodeoxycholate (not taurodeoxy-cholate) were detected.

Preparation of crude 'bile acid esters'. King penguincrude bile salts (1 g.) were hydrolysed at 1150 for 4 hr. in a

I957sealed metal bomb with 2-5N-NaOH (10 ml.). The acidsobtained by acidification ofthe diluted contents ofthe bombafter the addition of excess of sodium chloride were washedby manipulating the usually sticky mass under water.Esterification was effected by dissolving the crude acids,freed from excess of water by decantation, in cold ethanolcontaining 2% (v/v) H2SO4 and leaving overnight. Theproduct was isolated by ether extraction of the mixtureafter dilution with water. The ether was washed withaqueous NaHCO3 and water, dried (Na2SO4) and evapor-ated. Yield of 'crude esters', approx. 560 mg. Acidificationof the bicarbonate washings gave no unesterified acid.

Chromatography on aluminaSeparation of ethyl esters. 'Crude esters', prepared as

above, were dissolved in benzene (about 20 ml.) and thesolution was poured onto a column (diam. 14 mm.) of A1203.Fractions eluted by solvents of varying polarities wereexamined chromatographically on paper and otherwise asdescribed. Representative results for A1203 columns of thetwo types used are given in Tables 2 and 3. A1203 (S) columnscaused some loss of the esters, probably by hydrolysis.Paper chromatography of column fractions. Methods

previously described (Haslewood, 1954) were used. Frac-tions suspected of containing dihydroxy esters (eluted withethyl acetate or ether-benzene) were run in system A (Bush,1952). Later fractions were run in system B5 or, moresuccessfully, in system B3 (Bush, 1952). Spot 'X' (Tables 2and 3) ran at about 68% of the rate of ethyl cholate insystem B3. Combined fractions 31-35 from A120, (N)columns showed, in addition to spot 'X', a spot running insystem B3 at a rate slightly slower than that of 'X' and alsoa spot running at about 25% of the rate of ethyl cholate.

Identification of chenodeoxycholic acid. The gums obtainedin fractions 4 and 5 (Table 2) were combined and dissolved in50% (v/v) acetic acid-acetic anhydride (4 ml.); acetylationwas effected by adding, with cooling, 1 drop of 8-5N-perchloric acid. After 15 min. the mixture was diluted withwater and extracted with ether. Evaporation of the washedand dried (Na2SO4) ether left a gum which was purified byrunning it through A1203 (S, 5 g.) in benzene (10 ml.). Thebenzene-eluted material (226 mg.) crystallized from lightpetroleum in long colourless needles, m.p. 98-100°. Re-crystallization from light petroleum raised the m.p. to 103-1040, not depressed by admixture with authentic ethyldiacetyl chenodeoxycholate (m.p. 103-104°).

Identification of cholic acid. The partly crystalline materialfrom (combined) fractions 6-8 (Table 2) was washed with alittle 25% (v/v) ether-light petroleum, and the solid(93 mg.) obtained by filtration was recrystallized frombenzene-light petroleum to give needles of m.p. 1630, notdepressed by admixture with authentic ethyl cholate. Thisester (approx. 10 mg.) in cold acetic acid (2 ml.) was oxidizedby adding, with mixing, a few drops of 20% CrO3 at roomtemperature. The resultant dehydroester (recovered bydilution with water, extraction with ether and evaporationof the washed and dried extract) was recrystallized fromaqueous ethanol to give needles of m.p. 2130, not depressedby authentic ethyl dehydrocholate.

Evidence for the presence of tetrahydroxynorsterocholanicacid. In an early experiment, material eluted froman A1203 (N) column with 10% (v/v) ethanol-ether wasrecrystallized from ethyl acetate as fine needles, m.p. 174-1750 (Found: C, 71-3; H, 10-3%). This substance ran at the

324

KING PENGUIN BILE SALTSsame speed as ethyl cholate on a paper chromatogram insystem B5 (Bush, 1952) and had a 'fingerprint' (1200-900 cm.-') infrared spectrum almost identical with that ofethyl cholate. Hydrolysis followed by re-esterification withethanol of this substance gave a product which, aftercrystallization from benzene-light petroleum, had m.p.195-1980.In another experiment, combined fractions [500 mg.,

20% (v/v) acetone-ether eluate] from A1203 (N) columnswere left for 2 days with cold ether. The crystalline residueleft after decantation of ether was washed with cold ether;it then had m.p. 172-176°. This material was recrystallizedfrom aqueous ethanol, light petroleum-acetone (twice),aqueous ethanol and benzene (in that order), finally givingwhite needles (14 mg.) of m.p. 196-200° (Found: C, 71F5;H, 10-7. C26,H4405 requires C, 71-6; H, 10-1. C29H5008requires C, 70 5; H, 10-1 %).

This material (5 mg.) was dissolved by gentle warmingand left for 2 hr. in ethanol (0.3 ml.) with 5N-KOH (2 drops)and water (1 drop). The solution was diluted with water andtreated with excess of 5N-HCI and of NaCl. The gelatinousprecipitate was collected, washed with water and dissolvedin ethanol. Evaporation left a residue which was warmedwith ethyl acetate (about 1 ml.) and filtered after cooling.Repetition of this process finally removed a persistentamorphous impurity and allowed the preparation of a clearsolution in about 0 5 ml. of ethyl acetate. On standing, at

about 200 and then 50, this deposited the bulk ofthe productas white needles; solvent was decanted and the crystals werewashed with cold ethyl acetate. They then had m.p. 198-2000, depressed to 185-194° by a sample of cholic acid (m.p.196-1980), and showed the same behaviour after melting asKazuno's tetrahydroxynorsterocholanic acid (see below).In another experiment a similar hydrolysis of a sample ofcrystalline ester gave after treatment of the product withethyl acetate white needles of m.p. 211-214°, not depressedby Kazuno's acid (below) and showing the same behaviourafter melting as this substance.

Tetrahydroxynorsterocholanic acid, given by ProfessorT. Kazuno of Okayama Medical School, had m.p. 215-217°;at about this m.p. formation of a small amount of freshcrystals could be seen, and these decomposed at about 2980.Kazuno's acid (1-6 mg.) was left in solution for 2 days with2% (v/v) H2S04-ethanol (0.2 ml.). The mixture was treatedwith excess of aqueous NaHCO3 and the solid ester collectedand crystallized from dilute ethanol, giving white needles ofm.p. 200-203°, not depressed by the above ester, [m.p. 196-200°, prepared from the 20% (v/v) acetone-ether eluates ofA1203 (N) columns]. Both esters ran at the same rate asethyl cholate on paper in Bush's (1952) system B3; ethyl3x:7a: 12x-25D-trihydroxycoprostanate ran at more thantwice this rate.

Isolation of 'ketonic' fraction. A fraction (240 mg.)corresponding to 4-5 of Table 2 was boiled under reflux for

Table 2. Alumina chromatography (first type) of ethylated 'bile acids'from King penguin bile

The crude esters (1050 mg., black gum) were fractionated on 30 g. of A1203 (S).

Fractionno.1, 23456-89, 10

111213

Eluted by(100 ml. for

each fraction)BenzeneEtherEthyl acetateEthyl acetateEthyl acetateEthyl acetateEthyl acetateEthanolEthanol

Total eluted (see text)

wt.(mg.)1974

1012281575016

1022

857

AppearanceOilOilGum tGum )Partly cryst.SolidSolidSolidSolid

Results of paper chromatography(for methods, see text)

Spot as ethyl chenodeoxycholateSpot as ethyl cholate

Spot as ethyl cholate and spot 'X' (see text)

Table 3. Alumina chromatography (second type) of ethylated 'bile acids'from King penguin bile

The crude esters (731 mg.) were fractionated on 7-3 g. of A1203 (N) (see text).

Fraction Eluted byno. (solvent, ml. total vol.)1 Benzene, 1002-8 Benzene, 3509, 10 10% (v/v) ether-benzene, 150

11, 12 20% (v/v) ether-benzene, 15013, 14 50% (v/v) ether-benzene, 15015 Ether, 10016, 17 20% (v/v) acetone-ether, 20018-26 20% (v/v) acetone-ether, 45027-30 50% (v/v) acetone-ether, 20031, 32 Acetone, 10033, 34 10% (v/v) ethanol-acetone, 15035 25% (v/v) ethanol-acetone, 5036 Ethanol, 50

Total eluted

Wt.(mg.)316581912121

152484128318

Trace726

Appearance

Oilf

GumGumGum)Partly cryst.Gum iGumGumGumGum i

Results of paper chromatography(for methods, see text)

Spot as ethyl chenodeoxycholate

Spots as ethyl chenodeoxycholate andethyl cholate

Spot as ethyl cholate and (?) unknownsubstance

Spot as ethyl cholate and 'X' (see text)Spots as ethyl cholate, 'X' and at least 2other compounds (see text)

Vol. 67 325

I. G. ANDERSON, G. A. D. HASLEWOOD AND I. D. P. WOOTTON0 75 hr. with Girard's reagent T (100 mg.) in ethanol(3 0 ml.) with acetic acid (0.3 ml.). The oily 'ketones',isolated in the usual way, weighed 65 mg. This material inethanol (5 ml.) was warmed with 5N-KOH (0.5 ml.) for0 5 hr. Dilution with water and acidification with 2N-HClgave semisolid acids which were collected, washed and dis-solved in a minimal amount of ethyl acetate. After 9 dayssolvent was decanted from crystals: the latter (about 1 mg.)had m.p. 186-192°, and from aqueous ethanol gave colour-less crystals of m.p. 194-199°. These were methylated withethereal diazomethane, and the residue, after removal ofsolvent, was dissolved in CS2. In the range 1200-900 cm.-'the infrared spectrum resembled closely that of methyldeoxycholate (methyl 3a:12a-dihydroxycholanate) and intheregion 1800-1650 cm.-' only one bandwas present, whichwas attributed to the carboxylic acid ester. The nature ofthis substance, evidently carried over from the 'non-ketones', has not been further investigated.

Partition chromatographyAn attempt was made to separate ethyl cholate and ethyl

tetrahydroxynorsterocholanate and their chromic oxidationproducts on Celite columns as follows. Celite (kieselguhr,

I957Hyflo Supercel, Johns-Manville Co. Ltd.) was washed withacid and water as described by Carpenter & Hess (1956) andthen dried at 600. For each mixture solvent systems werefirst found in which the partition coefficient moving phase/stationary phase was about 0-2. The stationary phase wasmixed with the Celite and the whole transferred with excessof moving phase: the column was packed as described byHoward & Martin (1950). Substances to be separated weredissolved in not more than 0 7 ml. of stationary phase; thesolution was run on 0-1 g. ofdry Celite, lightly packed on thetop of the column as soon as supernatant moving phase haddrained from it. Carpenter & Hess's (1956) theoreticaltreatment enabled a prediction to be made of the volume(VE) needed to elute the desired compounds, and fractions of2 ml., embodying VB, were collected automatically intoweighed tubes. Table 4 gives details of columns; Carpenter& Hess's terms are used: Vs = vol. of stationary phase;Vl,p.=wt. (g.) of Celite/density of Celite (1.8); nrr2h = vol. ofcolumn. The results are shown in Table 5.

Preparation and chromatography of oxidation product(Celite columns 4 and 5 of Tables 4 and 5). The mixture ofethyl esters (11 mg., m.p. 178-200°) recovered from Celitecolumn no. 3 (Table 5) in acetic acid (0 4 ml.) was left with

Table 4. Details of partition column8 on Celite

For explanation of symbols see text. The parts by volume of solvents given were mixed and equilibrated in a separatingfunnel. The lower phase was then used as the stationary, and the upper as the moving, phase.

Celitecolumn

no.1

System(parts by vol.)

Heptane, 5: benzene, 5;ethanol, 10; water, 4

2 As col. no. 1

3 As col. no. 14 Light petroleum (b.p.

83-95°), 4; benzene, 1;ethanol, 5; water, 1

5 As col. no. 4

Substance put on column(mg. in ml. of stationary phase)Ethyl cholate (21 in 0 3)

King penguin ethyl esters,crystallized from benzeneto m.p. about 180-1960(17 in 0.3)

As above (17 in 0.3)Chromic oxidation productof the above (described intext; 10 in 0 7)As above (11 in 0 7)

Wt. ofCelite(g.)9

Vs(ml.)5

irr2 x h(cm.2 x cm.)1-29 x 16*4

Rateof flow(ml./hr.)

5*2

9 5 1F29 x 16'4 5-2

36 20 1-4 x 70 11.09 5 1F4 x 17-9 15-0

36 20 1-4 x 73 11.0

Table 5. Results ofpartition chromatography on Celite columns described in Table 4

Fractions of 2 ml. of effluent were collected.

Vol. (ml.)Celite of effluentcolumn (VE) giving

no. crystals1 24-34

2 22-34

Eluted crystals

Wt.(mg.)21

Approx. m.p.163-1640

Character of graph:mg. eluted/ml. VI/VBSingle sharp peak

17 178-2000 Single sharp peak

3 115-143 17 178-2000 Single sharp peak

4 42-5052-6062-66

5 176-208210-250

2-7 214-22703-8 140-21601-6 148-1580

(A) 7.3 180-2260(B) 3-0 156-1660

Sharp peak withlater tailing

Broad peak withconsiderable tailing

RemarksProbably the m.p. of pure ethylcholateAt peak, 2 ml. fraction containedabout 4 mg.At peak, 2 ml. fractions containedabout 2 mg.

For possible interpretation, seetext

No fraction contained more thanabout 0.5 mg. For furthertreatment of A and B, see text

326

KING PENGUIN BILE SALTS

20% CrO0 (0.1 ml.) for 15 min. Excess of water and thenNaCl were added, and the precipitate was extracted withether. The ether was washed with water, aqueous NH3 andwater, and dried (Na2SO4) and evaporated. The residue(9-8 mg.) had m.p. about 135-195'. Chromatography ofthis material gave results shown in Table 5; these are furtherdiscussed below.For the Celite column no. 4 (Table 5), if the 2-7 mg.

portion, m.p. 214-227', is taken as ethyl dehydrocholate, the1-6 mg. portion, m.p. 148-158°, as a substance which whenpure would melt at (say) about 1700, and the 3-8 mg., m.p.140-2160, as being composed of equal weights of the twocompounds, then the approximate composition of themixture recovered from the column was: ethyl dehydro-cholate(2-7 + 3.8/2 =4-6mg.), 57 %; substanceof m.p. about1700 (1.6 + 3.8/2 =3-5 mg.), 43 %.Eluted material was further treated as follows: Fractions

A (Celite column no. 5, Table 5) were combined and re-crystallized from aqueous ethanol, giving long colourlessneedles (4 mg.), m.p. 221-223°, not depressed by ethyl de-hydrocholate (m.p. 221-223°); fractions B, from the samecolumn, similarly treated, gave fine colourless needles(2 mg.)., m.p. 157-1660; these (1 mg.) were dissolved in CS2and the infrared spectrum of the solution in the range 1200-900 cm.-' was compared with that similarly obtained fromthe above specimen of ethyl dehydrocholate. There weredefinite differences in the spectra, in spite of an overallsimilarity.

DISCUSSION

Biological. The evolutionary position of penguinshas been fully discussed by Simpson (1946), and theconsensus of opinion appears to be that there are noadequate grounds for believing that the line ofdescent of these birds from reptiles differs from thatof the other Carinate birds. Our findings do notconflict with this view, for there is clearly a closeresemblance between the bile salts of the Kingpenguin and those of the other birds mentioned inTable 1. The only obviously different feature is theoccurrence in King penguin bile of tetrahydroxy-norsterocholanic acid and probably of cholic acid inamounts considerably greater than have beenfound in the bile of other birds. In preliminarywork we have found the same to be true for the bilesalts of the Gentoo penguin, Pygosceli8 papua. Thisdifference may have a dietary origin; however, wethink it desirable to defer further discussion of thebiological significance of the differences in bird bileacids until many more species have been examinedand the chemical nature of tetrahydroxynorstero-cholanic acid has been elucidated.

Chemical. The most striking chemical finding inthe present work is the great similarity betweencholic and tetrahydroxynorsterocholanic acids andtheir ethyl esters. Ohta's (1939b) tetrahydroxy-norsterocholanic acid was isolated from the bile ofa fish, and so presumably was that used by Isaka(1940). The sample sent to us by Professor Kazunowas, we believe, obtained from chicken bile byYamasaki (1951), who put forward the view that it is

identical with Ohta's acid. Our new material isundoubtedly substantially the same as, though lesspure than, Kazuno's sample. Our analyses suggestthat ethyl tetrahydroxynorsterocholanate mayhave the formula C2,H4405 : however, the analyticalsample certainly contained some ethyl cholate. Ifthe formula C26H4405 proves to be correct, theparent acid, C24H4005, is an isomer of cholic acid.Such isomerism could lie at an asymmetric centre orcentres, for example, at C-17 or C-20 in the steroidnucleus. The isomerism, if it exists, is not concernedonly with the nuclear hydroxyl groups (which webelieve on spectroscopic evidence to be at 3:7oc and12a: Haslewood & Wootton, 1956), for if it residedin these groups alone the product obtained byoxidation of 'ethyl tetrahydroxynorsterocholanate'with chromic acid would have been ethyl dehydro-cholate, instead of containing, as it did, a largeproportion of a quite different substance. We deferfurther discussion on the chemical nature of tetra-hydroxynorsterocholanic acid until more material isavailable. It is clear that King penguin bile alsocontains a number of hitherto unknown substances,which may be bile acids. Examples of these are:the substance giving spot 'X' (Table 2), the twocompounds mentioned in fractions 33-35 ofTable 3 and the non-ketonic substance finally givingan infrared spectrum like that of methyl deoxy-cholate, described on p. 326.

SUMMARY

1. The bile salts of the King penguin (Apteno-dite8 patagonica) have been shown to containchenodeoxycholic, cholic, tetrahydroxynorstero.cholanic and at least four other (unidentified) acids.The proportion of 'cholic-tetrahydroxynorstero-cholanic' acids was greater than that reported inthe bile of other birds. Apart from this, Kingpenguin bile salts resembled those of the otherbirds examined by previous workers.

2. An unsuccessful attempt has been made toseparate completely by crystallization and partitionchromatography the constituents of the 'ethylcholate-ethyl tetrahydroxynorsterocholanate 'mix-ture from Kingpenguin bile salts. The purest sampleof 'ethyl tetrahydroxynorsterocholanate' preparedby us from this mixture had m.p. 196-200°, gaveanalytical figures in agreement with C26H44O5, andwas hydrolysed to a mixture consisting largely of'tetrahydroxynorsterocholanic acid', apparentlyidentical with a sample earlier given to us byProfessor T. Kazuno. Ethyl cholate, C2,H440.,similarly recovered from partition chromatographyon Celite (kieselguhr) columns, had m.p. 163-164°.

3. Chromic oxidation of impure 'ethyl tetra-hydroxynorsterocholanate', m.p. 180}196', gavea mixture from which was obtained a specimen of

Vol. 67 327

328 I. G. ANDERSON, G. A. D. HASLEWOOD AND I. D. P. WOOTTON I957pure ethyl dehydrocholate, m.p. 221-223°, and animpure but different ester, m.p. 157-166°. The infra-red spectra of these two substances in the range1200-900 cm.-' showed an overall similarity butalso definite differences.

The authors offer their thanks to the following, withoutwhose generous help in collecting King penguin bile thiswork could not have been undertaken: Dr E. C. Appelbyand the Royal Zoological Society of Scotland, and BernardStonehouse of the Falkland Islands Dependencies Surveyand the Edward Grey Institute of Field Ornithology,Oxford.

REFERENCES

Bush, I. E. (1952). Biochem. J. 50, 370.Carpenter, F. H. & Hess, G. P. (1956). J. Amer. chem. Soc.

78, 3351.Haslewood, G. A. D. (1954). Biochem. J. 56, 581.Haslewood, G. A. D. (1956). Biochem. J. 62, 637.Haslewood, G. A. D. & Sjovall, J. (1954). Biochem. J. 57,

126.Haslewood, G. A. D. & Wootton, I. D. P. (1956). Biochem. J.

63, 3P.Haslewood, G. A. D. & Wootton, V. (1950). Biochem. J. 47,

584.

Hosizima, T., Takata, H., Uraki, Z. & Sibuya, S. (1930).J. Biochem., Tokyo, 12, 393.

Howard, G. A. & Martin, A. J. P. (1950). Biochem. J. 46,532.

Isaka, H. (1940). Hoppe-Seyl. Z. 266, 117.Ishihara, T. & Mori, T. (1938). Arb. med. Univ. Okayama, 5,

538.Maeda, K. (1938). Arb. med. Univ. Okayama, 6, 101.Marsson, Th. (1849a). Liebigs Ann. 72, 317.Marsson, Th. (1849b). Arch. Pharm., Berl., 58, 138.Ohta, K. (1939a). Arb. med. Univ. Okayama, 6, 193.Ohta, K. (1939 b). Hoppe-Seyl. Z. 259, 53.Ono, K. & Tamura, R. (1953). J. Jap. biochem. Soc. 25, 112.Shoppee, C. W. (1949). J. chem. Soc. p. 1671.Simpson, G. G. (1946). Bull. Amer. Mu&. nat. Hist. 87, 1.Sj6vall, J. (1955). Ark. Kemi, 8, 299.Takahashi, K. (1938). Hoppe-Seyl. Z. 255, 277.Wiggins, H. S. (1955a). Ph.D. Thesis: University of

London.Wiggins, H. S. (1955b). Biochem. J. 60, ix.Windaus, A., Bohne, A. & Schwarzkopf, E. (1924). Hoppe-

Seyl. Z. 140, 167.Wootton, I. D. P. & Wiggins, H. S. (1953). Biochem. J. 55,

292.Yamasaki, K. (1933). J. Biochem., Tokyo, 18, 323.Yamasaki, K. (1951). J. Biochem., Tokyo, 38, 93.Yonemura, S. (1926). J. Biochem., Tokyo, 6, 287; 8, 79.

The Effect of Sheep-Rumen Contents on Unsaturated Fatty Acids

BY F. B. SHORLAND, R. 0. WEENINK,Fat8 Re8earch Laboratory, Wellington

A. T. JOHNSPlant Chermi8try Laboratory, Palmer8ton North

AND I. R. C. McDONALDDominion Laboratory, Department of Scientific and Indu8trial Research, Wellington, New Zealand

(Received 19 March 1957)

Diets rich in unsaturated oils modify the depot fatsof most animals to resemble the dietary fats, butruminant depot fats are relatively unaffected (cf.Shorland, 1955). In addition, ruminant fats containhigher proportions of fully saturated glyceridesthan would be expected on the basis of the even orwidest distribution rule of Hilditch (1956). Otherpeculiarities ofruminant fats include the presence ofsubstantial amounts (5-10%) of tran8 unsaturatedacids (Hartman, Shorland & McDonald, 1954,1955) and ofmono- and di-unsaturated acids whichdiffer from oleic and linoleic acids in the position oftheir double bonds (cf. Shorland, 1956). Hilditch(1956) has suggested that the peculiarities of theglyceride structure of ruminant fats may be due tothe hydrogenation in 8itU of preformed oleoglycer-ides. However, while this process may possiblyoccur, the most important factor is probably the

hydrogenation of the dietary unsaturated fattyacids by the rumen micro-organisms, giving rise toless-unsaturated acids and stearic acid.

Perhaps the first observation of the effect of therumenon the dietary fatwasmade by Reiser (1951).He reported that linolenic acid present in linseed oilwas converted in vitro into linoleic acid by the actionof sheep-rumen contents. Similar results wereobtained by Hoflund, Holnberg & Sellman (1956),using fistulated sheep.As a result of the observation of Willey, Riggs,

Colby, Butler & Reiser (1952) that the depot fats ofsteers feed on cottonseed oil contained more stearicacid than the controls, R. Reiser (private communi-cation) suggested that the high content of stearicacid in the depot fats of ruminants was due to thehydrogenation of dietary C18 unsaturated acids inthe rumen. This suggestion was further supportedc