A More Detailed Study of Bile Salt Evolution, Including Techniques ...

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Biochem. J. (1974) 141, 485-494 485 Printed in Great Britain A More Detailed Study of Bile Salt Evolution, Including Techniques for Small-Scale Identification and their Application to Amphibian Biles By IAN G. ANDERSON,* GEOFFREY A. D. HASLEWOOD,* ROBERT S. OLDHAM,t BERNARD AMOSI and LASZLO TOKIS,l *Department of Biochemistry and Chemistry, Guy's Hospital Medical School, London SEI 9RT, U.K., tDepartment of Zoology, University of Ibadan, Nigeria, and $Institute of Organic Chemistry, Syntex Research, Palo Alto, Calif. 94304, U.S.A. (Received 13 March 1974) 1. Methods have been developed for the isolation and identification of small amounts of bile salts and of bile acids and alcohols obtained by solvolysis. These methods involve preparative and analytical t.l.c., purification on columns of protonated A1203 and Sephadex LH-20 and also g.l.c.-mass spectroscopy of solvolysis products. 2. Application to 29 species of frogs and toads has confirmed the constancy of bile salt patterns in a single species, including colour phases in two instances, and has revealed great variations between different species in some genera (e.g. Rana, Ptychadena) and little difference between widely distributed species in others (e.g. Bufo). 3. Taxonomic deductions should be made with caution and with regard to the physiological significance of the biochemical character considered. The molecular differences found might be interpreted as indicating variations in the rate of evolution. Studies of the bile salts of many vertebrate species have resulted in the almost complete elucidation of all chemical problems encountered and also in a concept of bile salt evolution that is both biochemically and taxonomically acceptable (Haslewood, 1971a). The circumstantial evidence derived from biosynthetic and species distribution studies suggests that evolution of the principal bile salts derived from cholesterol has taken the course shown in Scheme 1. One substance of type B (Scheme 1) has been isolated, from lamprey bile (Haslewood & Tokes, 1969); its biochemical affinities are unknown. It is uncertain whether substances of type C give rise to C24 acids, although this is possible (Danielsson & Kazuno, 1964). In many vertebrate groups (most teleosts, most lizards, snakes, birds and mammals) bile salt evolu- tion has proceeded to C24 acids, so that variations now to be seen comprise no more than differences in the pattern of hydroxylation of these substances. B C24 bile alcohol sulphates Cholesterol A C27 bile alcohol sulphates 1 C C26 and C25 bile alcohol sulphates D C2, bile acids, taurine conjugates E C24 bile acids, taurine conjugates F C24 bile acids, glyciiie conjugates Scheme 1. Apparent evolutionary relationships ofthe bile salts Vol. 141

Transcript of A More Detailed Study of Bile Salt Evolution, Including Techniques ...

Page 1: A More Detailed Study of Bile Salt Evolution, Including Techniques ...

Biochem. J. (1974) 141, 485-494 485Printed in Great Britain

A More Detailed Study of Bile Salt Evolution, Including Techniques forSmall-Scale Identification and their Application to Amphibian Biles

By IAN G. ANDERSON,* GEOFFREY A. D. HASLEWOOD,* ROBERT S. OLDHAM,tBERNARD AMOSI and LASZLO TOKIS,l

*Department of Biochemistry and Chemistry, Guy's Hospital Medical School, London SEI 9RT, U.K.,tDepartment of Zoology, University of Ibadan, Nigeria, and $Institute of Organic Chemistry, Syntex

Research, Palo Alto, Calif. 94304, U.S.A.

(Received 13 March 1974)

1. Methods have been developed for the isolation and identification of smallamounts of bile salts and of bile acids and alcohols obtained by solvolysis. Thesemethods involve preparative and analytical t.l.c., purification on columns of protonatedA1203 and Sephadex LH-20 and also g.l.c.-mass spectroscopy of solvolysis products.2. Application to 29 species of frogs and toads has confirmed the constancy of bile saltpatterns in a single species, including colour phases in two instances, and has revealedgreat variations between different species in some genera (e.g. Rana, Ptychadena) andlittle difference between widely distributed species in others (e.g. Bufo). 3. Taxonomicdeductions should be made with caution and with regard to the physiologicalsignificance of the biochemical character considered. The molecular differences foundmight be interpreted as indicating variations in the rate of evolution.

Studies of the bile salts of many vertebratespecies have resulted in the almost completeelucidation of all chemical problems encountered andalso in a concept of bile salt evolution that is bothbiochemically and taxonomically acceptable(Haslewood, 1971a). The circumstantial evidencederived from biosynthetic and species distributionstudies suggests that evolution of the principalbile salts derived from cholesterol has taken thecourse shown in Scheme 1. One substance of type B

(Scheme 1) has been isolated, from lamprey bile(Haslewood & Tokes, 1969); its biochemical affinitiesare unknown. It is uncertain whether substances oftype C give rise to C24 acids, although this is possible(Danielsson & Kazuno, 1964).

In many vertebrate groups (most teleosts, mostlizards, snakes, birds and mammals) bile salt evolu-tion has proceeded to C24 acids, so that variationsnow to be seen comprise no more than differencesin the pattern of hydroxylation of these substances.

B C24 bile alcoholsulphates

Cholesterol

A C27 bile alcohol sulphates

1 C C26 and C25 bile alcohol sulphates

D C2, bile acids, taurine conjugates

E C24 bile acids, taurine conjugates

F C24 bile acids, glyciiie conjugates

Scheme 1. Apparent evolutionary relationships ofthe bile salts

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In other groups, molecules of more primitive typesare found, apparently without C24 acids, suggestingthat the animals may have become evolutionarily'senescent' as far as their bile salts are concerned. Ina few animal groups, numerous small morphologicaland behavioural differences between closely relatedspecies suggest that perhaps evolution is very active;such groups can be found, for example, amongstfishes of the families Cyprinidae and Catostomidaeand also amongst anuran amphibians (frogs andtoads). It so happens that the bile salts both ofcyprinids and catostomids (Anderson & Haslewood,1970) and of anurans show large chemical variations.A few anuran species have already been examined(Haslewood, 1967; Kuramoto et al., 1973) and werefer to some of these below (see the Discussionsection).We have been fortunate in having the opportunity

to study the numerous frog and toad species livingin Nigeria, and we set out to identify the bilesalts of some of these animals and of other relatedanurans, with the purpose of assessing their value astaxonomic characters and as possible indicators ofevolutionary 'vigour' or 'senescence'.To carry out this work it was necessary to develop

new techniques, suitable for the identification ofsuch amounts of bile salts as were available from thefew small gall bladders that we could usually collect.We now describe these techniques, their applicationsto some amphibian biles, the results obtained and ourtentative assessment of their significance.

MAterials and Methods

General

Solvents (except analytical-quality acetic acid)for chromatography were redistilled at 4-6 weeklyintervals; methanol and ethanol were redistilled oversolid KOH. T.l.c. plates of silica gel were kept in asealed container and heated for 30min at 100°Cbefore use. For loading t.l.c. plates, adding solventsto powdered KBr for i.r. spectroscopy and for generaltransference of small amounts of liquid we used a100ul g.l.c. syringe (model 100 A-RN, ScientificGlass Engineering Pty. Ltd., Melbourne, Australia)which fitted into an Agla Micrometer Syringe Outfit(Burroughs Wellcome, London N.W.1, U.K.).

Bile salts

Nigerian amphibians were usually captured atnight at their aquatic breeding sites. They were keptfor periods varying between 1 h and 2 weeks, withoutfood, before being killed with MS 222 (Tricainemethansulphonate, Sandoz Ltd., Basle, Switzerland).The gall bladders were then immediately dissectedfree and placed in an excess of ethanol. Usually,gall bladders from several frogs were kept together

as a single sample for analysis. Most of the sampleswere drawn from single localities, but in a few cases(indicated as Western or Mid-Western Nigeria inTable 1) they were drawn from localities separatedby as much as 500 miles. For Arthroleptis and perhapsalso Hylarana there are behavioural indicationsthat the populations sampled, although appearingmorphologically homogeneous, may include morethan one species. Bile salts were partially purifiedas described by Haslewood (1967).

Lr. spectroscopy

Material (50-200,ug) in methanol (10,ul) was put onto powdered KBr (20-25mg) and dried at 40°C for atleast 15 min. The mixture was repowdered and madeinto a disc (diam. 1.5mm) in the usual way. Spectrawere taken with a model 137 or 157G spectrophoto-meter (Perkin-Elmer Co. Ltd., Beaconsfield, Bucks.,U.K.).

G.l.c.

This was as described by Anderson & Haslewood(1970). Retention times of trimethylsilyl ethers rela-tive to that of methyl trimethylsilyl cholate (1.00)on the column used were: 26-deoxy-26-nor-5a-ranol,0.63; 26-deoxy-26-nor-5fi-ranol, 0.71; 26-deoxy-5a-ranol, 0.77; 26-deoxy-5,B-ranol, 0.88; 27-deoxy-5a-cyprinol, 1.12; 27-deoxy-5fi-cyprinol, 1.25; 5a-ranol,1.40; 5a-bufol, 1.52; 5fi-ranol, 1.57; 5fl-bufol, 1.72;5a-cyprinol, 1.82; arapaimol-A, 1.93; 5fi-cyprinol,2.04. The values for 27-deoxy-5a-cyprinol and26-deoxy-5a-ranol were deduced from the 5a-5f,differences determined experimentally in other cases.

G.l.c.-mass spectrometry

The solvolysis products (0.5-2.0mg) were silylatedwith an excess of N-trimethylsilylimidazole (PierceChemical Co., Rockford, Ill., U.S.A.) at 80°C for1-2h. Portions (1-2p1) of the silylation mixture wereanalysed directly on a Hewlett-Packard F and M-402gas chromatograph (Hewlett-Packard, Palo Alto,Calif., U.S.A.) which was interfaced, via an all-glassdouble-stage Biemann-Watson-type molecularseparator (Varian-MAT G.m.b.H., Bremen, Ger-many), with a Varian-MAT CH-7 mass spectrometer(Varian-MAT G.m.b.H.). When the presence of abile acid was detected, the analysis was repeated on afresh sample which was first methylated withdiazomethane and then silylated as above.The g.l.c. separations were carried out on a

column [1m; 3% OV-17 (Applied Science Labora-tories Inc., State College, Penn., U.S.A.) or 1.2m;3.8% UCW-98 (Hewlett-Packard)] at 235-250°C and50ml/min helium flow rate. The g.l.c.-mass spectro-meter interface and molecular separator temperatureswere kept at 240-250'C. The mass spectra were

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measured at 70eV ionizing voltage and at 1800Cion-source temperature. The data collection, unitresolution, mass identification and spectral plottingwere carried out on an IBM-1800 computer (IBMCorp., San Francisco, Calif., U.S.A.) equipped with24K core memory and an IBM Calcomp plotter.

Preparative and analytical t.l.c. for conjugates

Silica gels (Kieselgel G, from E. Merck A.-G.,Darmstadt, Germany; MN-Kieselgel G-HR fromMachery, Nagel and Co., Diiren, Germany andSilica gel CT from Reeve, Angel Scientific Ltd.,London E.C.4, U.K.) were tried for preparative t.l.c.in layers (0.5mm thick) on glass plates (5cm x 20cm).All proved satisfactory for periods up to 4 weeksand then appeared to 'age', subsequently givingpoorer separations. Precoated plates (0.25mm thick-ness, without indicator; E. Merck A.-G.) did not havethis disadvantage and gave less residue after elutionwith methanol.

Bile salts (up to 1mg) in methanol (50ul) wereloaded in as narrow and even a band as possibleat about 1.5cm from one end of the plate.After equilibration in the usual way for 15min, thejar containing the plate was tilted so that solventreached the silica gel. Chromatography was stoppedwhen the front had travelled about 18cm.

Solvent mixtures were chloroform-methanol-aqueous NH3 (sp. gr. 0.88, analytical grade), 15: 10: 1,by vol., or ethyl acetate-acetic acid-water, 15:12:2,by vol. After being dried at room temperature andthen at 100°C, the cooled plates were exposed toiodine vapour, the defined zones were scraped offandmaterial was eluted from the silica gel with methanol.The filtered methanolic extracts were evaporated todryness in a stream ofN2 and the residues washed witha little cold ether, redried and weighed. They were thendissolved in methanol (501l) and analysed by t.l.c.as previously described (Haslewood, 1971b) with themixture, acetic acid-3-methylbutyl acetate-water(7.5:7.5:3 or 9:7:3, by vol.) Standards were(systematic names of steroids are given in the legendto Table 1) purified sodium 5f,-bufol sulphate, bilesalts of Rana esculenta (mainly 5,6-cyprinol sulphate),Rana pipiens (mainly 5f8-ranol sulphate) and Disco-glossus pictus (mainly taurotrihydroxycoprostanate).Preparative t.l.c. with chloroform-methanol-aq.NH3, as described, separated the three substancesused as standards and also separated 5/8-cyprinolsulphate from 27-deoxy-5fi-cyprinol sulphate and5fi-ranol sulphate from 26-deoxy-5fi-ranol sulphate.It did not separate the 5c from the correspond-ing 5,B sulphates, nor did it separate the cyprinolfrom the bufol sulphates.

Analytical t.l.c. did not differentiate betweentaurotrihydroxycoprostanate and cyprinol sulphate;otherwise it had about the same discriminatory power

Vol. 141

as preparative t.l.c. Nevertheless, 3-methylbutylacetate-acetic acid-water mixtures were, in ourhands, better able to separate taurine conjugatesand bile alcohol sulphates than other solventsystems described in the literature.

Purification of conjugates on columns

(a) On protonated alumina. Small colums of pro-tonated alumina (A1203-HCI, 0.1 g) were used asdescribed by Haslewood (1971c); they were con-veniently packed by using capillary pipettes totransfer A1203-HCI suspended in methanol.Conjugates, eluted with methanol from silica gelafter preparative t.l.c., were used as the anions onA1203-HCI. After elution from A1203-HCl withammoniacal methanol (Haslewood, 1971c) theproducts were obtained as follows: (i) theNH3-water-methanol eluate was evaporated to dryness on aboiling-water bath in a N2 stream; (ii) excess ofaqueous 0.1M-Na2CO3 was added [for the A1203-HCI sample described by Haslewood (1971c), thiswas 2ml of 0.01M-Na2CO3] followed by excess ofethanol and evaporation to dryness was carried out asbefore; (iii) to the residue was added excess of 0.1 M-aqueous HCI (0.4ml for 2ml of 0.01 M-Na2CO3 usedabove) followed by ethanol (excess) and the mixturewas evaporated as before; (iv) the residue of NaCland sodium salts of conjugates was extracted withethanol (1-2ml) and the filtered extract evaporated.Repetition of this process (iv) gave the conjugatesalmost free from chloride and showing little impurityon i.r. spectroscopy, especially when precoatedsilica-gel plates had been used for preparative t.l.c.This method, involving contact with high concen-trations of protons, might be expected to causedecomposition in some cases and, althoughwe did notnotice this in the present work, the results should bechecked against the original bile salts.

(b) On SephadexLLH-20. This column, described byHaslewood & Haslewood (1972), gave conjugatesfree from major impurities. However, elution of'blank' columns with chloroform-methanol (1:1,v/v) gave material (0.01-0.02mg/ml of effluent)that showed very intense absorption in the region1500-1800cm-1; hence i.r. spectra of conjugatespurified by this method had to be interpreted withcaution. The region 625-1400cmni, containing bandsfrequently diagnostic of steroid nuclear hydroxylsubstitution patterns and configuration at C-5(Haslewood, 1967), were not affected.

Solvolysis of sulphates (cf. Burstein & Liebermann,1958)

Conjugates (1-4mg) were dissolved, with gentlewarming, in 0.1M-HCI (1 ml). NaCl (200mg) wasadded and dissolved; this usually produced turbidity

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Table 1. Bile salts ofsome anurans

The bile salts detected are labelled A-R, which arethefollowing: A, 5a-bufol sulphate [5a-cholestan-3a,7a,12a,25c,26-pentol(probably) 26 sulphate, formula I]; B, 5,8-bufol sulphate (the 513 epimer of compound A); C, 5a-cyprinol sulphate(5a-cholestan-3a,7a,12a,26,27-pentol 26 or 27 sulphate, formula II); D, 5,B-cyprinol sulphate (the 513 epimer of compoundC); E, 5a-ranol sulphate (5a-27-norcholestan-3a,7a,12a, 244,26-pentol 24 sulphate, formula III); F, 5,B-ranol sulphate (the513 epimer of compound E); G, 27-deoxy-5a-cyprinol (= 25-deoxy-5a-bufol) 26 sulphate; H, the 5,B epimer of compoundG; I, 26-deoxy-5a-ranol sulphate; J, the 5/I epimer ofcompound I; K, 26-deoxy-26-nor-5Sa-ranol, formula IV (sulphate); L,the 5/I epimer ofcompound K; M, taurotrihydroxycoprostanate [taurine-conjugated 3a,7ca,12a-trihydroxy-5,8-cholestan-26 (or 27)-oic acid, formula V]; N, unconjugated 3a,7a,12a-trihydroxy-5,8-cholestan-26 (or 27)-oic acid; 0, trihydroxy-bufosterocholenic acid (3a,7a,12a-trihydroxy-5,8-cholest-22-ene-24-carboxylic acid); P, unconjugated cholic acid; Q,arapaimol-A (5/8,25ct-cholestane-2,8,3a,7a,12a,27-pentol, formula VI); R, partially identified or unidentified steroids. Theevidence for identification was obtained by methods a-e which are as follows: a, analytical t.l.c. with or without preparativet.l.c.; b, i.r. spectroscopy of conjugate; c, g.l.c. of trimethylsilyl bile alcohols after solvolysis; d, g.l.c.-mass spectroscopy oftrimethylsilyl bile alcohols or trimethylsilyl ester ethers ofacids or methyl ester trimethylsilyl ethers of acids, after solvolysis;e, t.l.c. and i.r. spectroscopy of bile alcohols or acids after solvolysis. X, Chief bile salt; x, minor constituent; n.t., notspecifically tested for unconjugated acids.

Number of gallbladders in collec-

Family and tion (whereknown)species and origin

DiscoglossidaeDiscoglossus 28, Maltapictus

Pipidae 15, Witwatersrand,Xenopus laevis South Africa

6, IbadanWestern Nigeria

BufonidaeBufo b.formosus Japan

B. b. vulgaris 1, Oxford,England

B. maculatus 12, IbadanWestern Nigeria

B. marinus

B. regularis

Ranidae,Arthrolepinae

Arthroleptis(poecilonotus?)

Cardioglossaleucomystax

C. nigromaculata

Ranidae,CorniferinaeHylaranaalbolabris

Jamaica

Ibadan,Western Nigeria

24 Western andmid-westernNigeria

7, Ijebu-Ode,Western Nigeria

2, Ijebu-Ode,Western Nigeria

2, WesternNigeria

Bile salts. >~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.

A B C

x xd d

XabcdXabcd

xabcdXabcdXabcdxabcdXabcd

D E F G H I

xd

xd

x xd d

x xd cd

x Xcd cd

xcd

Xcd

x

cd

x X X x X xd cd ab ad cd cd

cdx x Xd cd cd

X Xcd cd

X xab a

Xad

Xad

xa

xa

xa

xa

x Xd ab

n.t.a

n.t.a

x Xd ac

d

X. mulleri

J K L M N O P Q R

x

ad

x* xd d

xd

xd

X Xtcd ab

cdXtab

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Table -continued

Number of gallbladders in collec-

Family and tion (whereknown),species and origin A

Ranidae, Raninae,Aubria 5, Westernsubsigillata Nigeria (forest)

Dicroglossus 9, Ibadan,occipitalis Western Nigeria

(savanna)Ptychadena 4 and 9,aequiplicata Western Nigeria

(two collections, (forest)examinedseparately)

P. longirostris 4 and 10, Western(two collections, and mid-westernexamined Nigeria (forest)separately)

P. maccarthyensis 3, Northern andEastern Nigeria(savanna)

P. mascareniensis Ibadan, Western xNigeria (forest) d

11, Western andMid-WesternNigeria (savanna)

3, Ibadan, X

Western Nigeria cd(savanna)

20, Commercialsource (Europe)

4, Ibadan, Western x

Nigeria(savanna) d6, Commercial

source (NorthAmerica)

5, Kent,England

R. temporaria Commercialsource (Europe)

RhacophoridaeHyperolius 13, 8 and 7concolor respectively,ibadensis Western Nigeria(colour phases (forest-savanna)J1, J2 and Fexaminedseparately)

H.fusciventris 20 and 8burtoni respectively(colourphases Jl Western Nigeriaand F examined (forest)separately)

Vol. 141

B C D E

x Xd ab

cdx xd cd

x Xd ab

cd

x Xd ab

cdx Xxd ab cd

cdx Xd ab

cdXcd

xcd

X

cd

X

abcd

X

abcd

X

abcd

Bile salts)

F G H I J K

X Xab abcd cd

x X x xd ab d cd

cdx x X x Xd c cd cd ab

cd

X x Xcd cd cd

L M N O P Q R

x xcd a

x x x

d ab adxd

xd

xd

X x Xab cd cdcd

x x X xd d cd cd

X xcd cd

x X

d cd d

X

ad

n.t.a

xd

xd

xcd

x

cd

X

abcde

x X

d abcd

x X

d abcd

xabcd

X

abcd

X

cd

xd

x x x

d d d

X

abe

Xtd

x x$d d

P. oxyrhynchus

P. taenioscelis

Rana esculenta

R. galamensis

R. pipienscomplex

R. ridibunda

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Table 1-continued

Number of gallbladders in collec-

Family and tion (where known)species

H. guttulatus(a mixture of allcolour phasesand phase JI ex-

Bile salts

and origin A B C D E F G H I J K L M N 0 P Q R4 and 7 respec- x X x X x x$

tively, Ibadan, d ab d ab d dWestern Nigeria cd cd(forest)

amined separately)H. nitidulus 4, Ilorin,

Western Nigeria(savanna)

xabcd

xabcd

x X$d d

* This remarkable acid was found by method d only in the bile of Bufo b. formosus, from which it had been previouslyisolated by Japanese workers and finally identified by Hoshita et al. (1967). We now confirm their structural assignment.

t The bile salts of two species of Cardioglossa appeared quite similar and are discussed in the Results section.t Hyperolius samples proved difficult to investigate by g.l.c. probably because of persistent impurities. A number of

constituents remain unidentified. The principal unknown substances in H. concolor ibadensis and H. nitidulus wereapparently identical.

HO

(I) (IV)

HO \CH20-CH0-S3-

HO

(V)

H(III) (VI)

ora precipitate. Ethyl acetate (4ml) was added and, thestoppered mixture was kept, with occasional mixing,at 37-38°C for 48 h. It was then extracted twice withethyl acetate. The aqueous portion was tested with

0.5M-BaCI2 (0.4ml) for SO42-. The ethyl acetate waswashed with water, -aqueous NaHCO3 solution(excess) and water, dried over Na2SO4 andevaporated, leaving neutral material (bile alcohols,

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etc.) as residue. Solvolysis appeared to be almostquantitative; the method caused some acetylation ofthe bile alcohols, which was readily detected byg.l.c.-mass spectroscopy. Unconjugated bile acidswere not fully removed by this procedure andappeared in the g.l.c.-mass spectra: of course theywere retained by omitting the bicarbonate washing.

Results

These are shown in Table 1. The assessment ofwhich were 'chief' bile salts was based on the originalt.l.c. and i.r. examination or on subsequentg.l.c. after solvolysis. In most cases, it was easyto decide which were the principal constituents,but in some of the more complex bile salts thisjudgement must be regarded as tentative. No freebile alcohols were detected by t.l.c. in any biles, butsome of the minor constituents seen on g.l.c. aftersolvolysis might have existed in the free form in theoriginal bile salts. Unconjugated bile acids werepresent in a number of cases; the condition ofcollection and storage were such as to make it certainthat these could not have arisen by post-mortemhydrolysis.A few hitherto undiscovered compounds were

detected and are chief bile salts in Cardioglossaleucomystax. A principal alcohol (present asmonosulphate) in this animal is apparently arapaimol-A (Haslewood & Tokes, 1972). The relative retentiontime of the maximum of the other chief g.l.c. peakgiven by the trimethylsilyl-solvolysed bile salts mightcorrespond to that of the 5a epimer of arapaimol-A,but mass spectroscopy shows that a mixture of sub-stances is responsible for this peak. Arapaimol-Awas also found as a minor constituent in three speciesof Hyperolius.A previously unrecognized substance is 26-deoxy-

26-nor-5f6-ranol detected, together with minoramounts of its 5a epimer (formula IV), in a few bilesand sufficiently predominant in Rana pipiens tosuggest that its isolation in substance might befeasible.

Apart from the above-mentioned compounds, thechief bile salts found were the bufol sulphates(5d, formula I), cyprinol sulphates (Sd, formula II)and ranol sulphates (5d, formula III), their C-terminaldeoxy derivatives and taurotrihydroxycoprostanate(formula V). Some bile salts, e.g. those of Ranaesculenta, R. galamensis and Discoglossus pictusshowed on t.l.c. faint spots corresponding inmobility to the taurine conjugate of cholic acid(3a,7a,12a-trihydroxy-5#-cholan-24-oic acid) orits 5a epimer. The amount of material concemedwas too small for isolation or preparative t.l.c. and(for the reasons cited in the Discussion section) wehave not attempted a complete analysis of the minorcojugated bile acids,Vol. 141

Discussion

MethodologyIdentification of conjugates depends on ability

to separate them; this is limited at present by thediscriminatory power of preparative t.l.c. I.r.-spectroscopic characterization of 5ca- or 5/1-3 a,7a, 12a-trihydroxy conjugates was convincing and, inaddition, we observed that the strong broad sulphateband in the region 1150-1300cm-I is almost smoothwith an -0 S03- group, but with the grouping-CH2 * S03- (taurine conjugates) there are well-marked shoulders at about 1175 and 1255cm-'.This feature enabled us to judge with confidencewhether conjugates consisted wholly or mainly ofsulphate esters or taurine conjugates, even whenimpurities obscured the amide bands at about (max.)1545 and 1650cm'1. We also noticed that ranol or26-deoxyranol sulphates showed a well-marked bandat about (max.) 946cm-' (5a) or 926cm-' (5,B).This we attribute to interaction between the-O S03- group at C-24 and the hydroxyl groupat C-12. Molecular models of ranol sulphates showthat these groups are in close proximity and caneasily be made to touch.The evidence for conjugates that we now present

is not as good as that for bile alcohols. Methods forhydrolysingconjugates ofC27 acids require prolongedheating in concentrated alkali at quite high tempera-tures and these conditions certainly cause somedecomposition. We have therefore postponed com-plete investigation of the bile acids in the hope thatmilder methods of hydrolysing the conjugates willbecome available.

G.l.c. was checked in two laboratories and wethink that the combined g.l.c.-mass-spectral evi-dence for alcohols is excellent. Unconjugated acidsin substantial proportions of the bile salts wereeasily identified by t.l.c. and g.l.c.-mass spectro-scopy. In summary, we believe that only minoramounts of conjugated bile acids have escapeddetection in this study.

All bile alcohols and acids listed in Table 1exhibited characteristic fragmentation patterns intheir mass spectra and could be identified thereforeeven in unresolved g.l.c. peaks. The mass values andrelative intensities of the fragment ions resultingfrom the sequential losses of the C-17 side chain andtrimethylsilyl alcohols from the steroid nucleiprovided clear distinction between the usual C-5epimeric 3a,7a,12ac-trihydroxy(m/e 253, 343 and 433ions) and 211,3a,7cc,12a-tetrahydroxy(m/e 251, 341,431 and 521 ions) skeletons. These considerationsalone could be misleading, however, with compoundshaving (or forming easily) a double bond in the sidechain. In these compounds, depending on theposition of the double bond, the loss of the sidechain is often associated with the transfer of two

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hydrogens to the expelled radical (Wyllie &Djerassi, 1968). The fragmentation patterns couldnot differentiate between certain C-3 epimericcompounds, e.g. 5a-cyprinol and its 3,8 isomer,latimerol.The size and oxidation pattern of the side chains

were evident from the high mass range of the spectra.The fully silylated compounds exhibited mostlyvery weak molecular ions with stronger peaks owingto the sequential losses of trimethylsilyl alcohols.Characteristic fragment ions can dominate thespectra in the presence of silyloxy groups along theside chain owing to preferential cleavage of acarbon-carbon bond adjacent to the silyloxy groups.These diagnostic cleavages can lead to chargedfragments in both high and low mass range of thespectra.

Biological

The already known bile salts found in this study(taurotrihydroxycoprostanate and themonosulphatesof the bufols, ranols and cyprinols and theirC-terminal deoxy derivatives) are those previouslyfound in amphibian biles. Kuramato et al. (1973)examined bile salts of Xenopus laevis, Bombinaorientalis, Hyla arborea japonica and three speciesof Rana. They carried out a dioxan-trichloroaceticacid solvolysis followed by alkaline hydrolysis andthen separated the product into neutral andacidic substances. These were separated by t.l.c. andsubsequently analysed by g.l.c. and mass spectro-metry. These methods are adequate for the identifi-cation of the main bile salts (although we have foundthat dioxan-trichloroacetic acid can cause somedecomposition in certain cases) and included anexamination for minor amounts of acids which we,for the reasons given above, did not undertake.However, it is unlikely that Kuramato et al. (1973)would have detected the large variety of alcoholsfound by our direct g.l.c.-mass spectroscopic analy-sis of the solvolysis products. Kuramato et al. (1973)found 5fi-cyprinol sulphate and taurotrihydroxy-coprostanate, as we did, as the chief bile salts inXenopus laevis and also small amounts of cholicacid and chenodeoxycholic acid (3a,7a-dihydroxy-5f8-cholan-24-oic acid). In other anurans (none ofwhich we have examined) they detected taurotri-hydroxycoprostanate, 5fl-bufol, 5a- and 5f6-cyprinol,5.8-ranol and small amounts of C24 acids. Aremarkable finding was that the bile salts of Bombinaorientalis consisted only of unconjugated trihydroxy-coprostanic acid and its C-24 hydroxylated derivative(varanic acid or an isomer), previously found innature only in lizards of the family Varanidae and inHeloderma horridum (Haslewood, 1967), but a knownintermediate in the biosynthesis of cholic acid fromcholesterol.

The few urodeles (newts and salamanders) pre-viously examined have biles like those of anurans(except of course those of Bombina orientalis) andthese show resemblances to bile salts of Latimeriachalumnae and the lungfishes, which are believed tobe the livingforms closest to the fish stocks ancestral toanurans and urodeles. It seems unlikely thatelucidation of the chemistry of the unidentifiedcompounds, for example in Cardioglossa, will muchalter this picture. Nothing so far discovered inamphibian bile supports the idea of a separatedescent of urodeles and anurans from ancestral fish.Our new methodology has enabled us to test afresh

the variations shown by bile salts in a single species.The examination of two separate collections ofPtychadena aequiplicatus and P. longirostris, threecolour phases of Hyperolius concolor and two suchphases of H. fusciventris (Table 1) all showed thateven in animal groups in which there are considerablevariations of bile salts, the pattern in a single speciesexhibits no more than quite small quantitativedifferences. The relevant g.l.c. 'profiles' in the casescited above were almost identical. These observationsstrengthen still further the original hypothesis of ourbile salt studies, that the primary bile salt chemistryis species-specific and genetically determined. Thereare no grounds for suspecting the presence of secon-dary bile salts (i.e. those modified by intestinalmicro-organisms) in amphibian bile.We were surprised to find that one species,

Ptychadena aequiplicata, had as its chief bile saltsthe monosulphates of C27- and C26-tetrols (27-deoxy-5fl-cyprinol and 26-deoxy-5fi-ranol). We had pre-viously thought, and observed, that for C26 and C27compounds at least five hydroxyl groups wererequired for a physiologically adequate bile salt.On morphological grounds the Pipidae and Disco-

glossidae are thought to be the two most primitiveof the families represented in our sample and alsoto be closely related. There is a basically primitiveskeletal structure but extant species are variouslyspecialized, often in a manner which can be corre-lated with their current niche. Discoglossus andXenopus both have a predominantly aquatic mode oflife and show structural physiological and behaviouralspecializations associated with this. In view of the'advanced' chemical structure of the bile salts of thesetwo families (Scheme 1) it would appear that the bilesalts represent a specialized aspect of their biologies.We are unable, however, to correlate this with theirfeeding habits. Although two other species whichhave predominantly aquatic habitat, namely Dicro-glossus occipitalis and Rana ridibunda, share withXenopus the bile salt taurotrihydroxycoprostanate, soalso does Arthroleptis, the most terrestrially adaptedspecies in our sample. In terms of diet the Pipidae areprobably the most distinctive members of oursample because, unlike those of the other species,

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the tadpoles are planktonic feeders, whereas theadults, also unlike the other species, feed entirely inwater. However, apart from taurotrihydroxycopro-stanate, the chief bile salt is 5,B-cyprinol sulphateand this is widespread throughout the series. Eventhe combination of the two compounds is notunique (Table 1).The genus Bufo comprises a widespread group of

anurans whose biology and evolution has beenthe subject of a recent comprehensive review (Blair,1972). Although widespread the genus is relativelyhomogeneous, its members showing clear resemb-lances in morphology, ethology and ecology. Severalof the species are genetically compatible. The bilesalts reflect this homogeneity (Table 1).

In theRanidae our three samplesfrom the subfamilyArthroleptinae are all small forest species withspecialized life cycles. They lay heavily yolkedeggs and in one of them, Arthroleptis, developmentis direct, there being no free-living tadpole stage. Inall three the bile salts are also specialized, either intheir structure or diversity.Our single example of the subfamily Corniferinae

is similar to most ranids in morphology and breedingbiology but differs in its arboreal habitat and associ-ated digital modifications. The bile salts are alsosimilar to those of some of the Raninae.Amongst our examples of the Raninae, Aubria

and Dicroglossus are both large predominantlyaquatic frogs, the former a forest and the latter asavanna species. Both have distinctive bile saltcombinations.The genus Ptychadena differs from Rana mainly

in the structure of the pectoral girdle. In other aspectsof their biology the two genera are basically verysimilar. The members of both, however, display aseries of specific variations, particularly in terms ofhabitat adaptations and behavioural isolatingmechanisms, which separate them ecologically andgenetically and which suggest that there has beenrecent evolution. A good example of this is theNorth American 'Rana pipiens' complex, which hasrecently been shown to consist of at least fourbiological species, almost indistinguishable morpho-logically (Littlejohn & Oldham, 1968). Intraspecificvariation is also striking in populations ofsome of thespecies; colour polymorphism in Ptychadena mas-careniensis may be cited as an example. The rangeof bile salt variation in the two genera is quitestriking. Of the 11 examples in our sample almostevery one is distinctive, although there are clearbiochemical relationships within the genus Ptycha-dena. As noted above, this variation does not extendto the population level, for separate samples ofP. aequiplicata and P. longirostris showed identicalbile salts.Our small sample of four African species from

the Rhacophoridae all belong to a genus which Schi0tz

Vol. 141

(1967), on grounds similar to those cited above,suggests is in a stage of rapid current evolution.Marked colour polymorphism exists in all fourspecies. They are adapted to arboreal life and havehabitats ranging from forest to savanna. The fourspecies show a broadly similar bile salt pattern butall have unidentified components and three containarapaimol-A. Clearly data are needed for moremembers of the family but on the basis of our smallsample it appears that the genus may showcharacteristics between those of the stable Bufoand variable Ptychadena and Rana.

It is difficult to compare anura at the family levelbecause it is not understood to what extent theRanidae, for instance, are polyphyletic. At thelevel of genus there is disagreement as to thegroupings and some authors (for example Inger,1968) group Dicroglossus, Hyla and Ptychadena asRana. The system used in Table 1 has the advantagefor our present purposes of grouping together frogswhich show clear morphological resemblances. Themost striking feature of our findings is the range ofinterspecific variability in the different genera. Thebile salts in some genera (e.g. Bufo) seem to varylittle and in others (Rana, Ptychadena) show largeinterspecific differences.These results may indicate that the existing

phylogenetic classification, particularly in the familyRanidae, is erroneous, or the observed differencesmay be a reflexion of differences in the rates of bilesalt evolution; in some genera such as Bufo this mayhave slowed or ceased, whereas in others such asRana it may still be very active.

Unfortunately the aspect of anuran physiologymost relevant to bile salt chemistry, namely the diet,is little investigated, although in a general senseit probably varies in relation to the habitat. It is alsopossible that the larval diet is the important variable,but on this there is no information. Although, ingeneral, 'modernization' of the bile salts goes hand inhand with advances in the level of organizationof the phenotype, the rate of this process must beaffected by the problems of fat digestion and absorp-tion that have to be solved and cannot be expected tobe the same in every animal group. If our results doindeed indicate that bile salt evolution is more activein some genera than in others, any taxonomic inter-pretation of this kind of biochemical evidence mustobviously be made with caution. For example,the bile salts of Discoglossus pictus (mainly taurotri-hydroxycoprostanate) are biochemically more ad-vanced than those of Rana esculenta (chiefly 5,B-cyprinol sulphate) (Scheme 1). However, it would notbe justifiable to conclude from this fact that thewhole phenotype of the former frog is more advancedthan that of the latter. Each biochemical characterto be used in taxonomy must be considered on itsmerits, having regard to the selective forces

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affecting its rate of evolution. It is our opinion thatthe evidence afforded by studies of bile salts, and thesuggestions about rates of evolution, will be valuablefor evolutionary and taxonomic studies whenconsidered in conjunction with other taxonomicparameters. Wallace et al. (1971, 1973) examined theserum albumins, fibrinopeptides, haemoglobins andDNA of frogs of the genus Rana and found unexpec-tedly large differences between species. Their generalfinding of great molecular diversity is fully supportedby bile salt studies. If, however, the bile salt chemistrydoes indeed indicate rapid evolution in some generabut not in others, it is difficult to understand howany time-scale can be applied to molecular differences,as suggested by Wallace and his colleagues.We express our gratitude to the following for collec-

tions of bile: Dr. P. G. D. Dean for Bufo b. vulgaris,Dr. T. Kazuno for Bufo b. formosus, Dr. S. Morrisseyfor Discoglossuspictus, Dr. A. U. Ogan for Bufo regularisand members of the Chemistry Department, Witwaters-rand University, South Africa, for Xenopus laevis.This is contribution no. 437 from the Institute ofOrganic Chemistry, Syntex Research.

ReferencesAnderson, I. G. & Haslewood, G. A. D. (1970) Biochem. J.

116, 581-587Blair, W. F. (1972) Evolution in the genus Bufo, University

of Texas Press, Austin, Texas

Burstein, S. & Lieberman, S. (1958) J. Biol. Chem. 233,331-335

Danielsson, H. & Kazuno, T. (1964) Acta Chem. Scand.18, 1157-1163

Haslewood, G. A. D. (1967) Bile Salts, pp. 26, 34-36, 87,90, Methuen and Co., London

Haslewood, G. A. D. (1971a) in Biochemical Evolutionand the Origin ofLife (Schoffeniels, E., ed.), pp. 191-202,North-Holland Publishing Co., Amsterdam

Haslewood, G. A. D. (1971b) Biochem. J. 123, 15-18Haslewood, G. A. D. (1971c) Biochem. J. 126, 27P-28PHaslewood, E. S. & Haslewood, G. A. D. (1972) Biochem.

J. 130, 89PHaslewood, G. A. D. & Tok6s, L. (1969) Biochem. J.

114, 179-184Haslewood, G. A. D. & Tokes, L. (1972) Biochem. J. 126,

1161-1170Hoshita, T., Okuda, K. & Kazuno, T. (1967) J. Biochem.

(Tokyo) 61, 756-759Inger, R. F. (1968) Explor. Parc. Nat. Garamba Miss. H.

de Saeger 52, 1-190Kuramoto, T., Kikuchi, H., Sanemori, H. & Hoshita, T.

(1973) Chem. Pharm. Bull. 21, 952-959Littlejohn, M. J. & Oldham, R. S. (1968) Science 162,

1003-1005Schi0tz, A. (1967) SpoliaZool. Mus. Haun. 25,1-346Wallace, D. G., Maxson, L. R. & Wilson, A. C. (1971)Proc Nat. Acad. Sci. U.S. 68, 3127-3129

Wallace, D. G., King, M.-C. & Wilson, A. C. (1973)Syst. Zool. 22, 1-13

Wyllie, S. G. & Djerassi, C. (1968) J. Org. Chem. 33,305-315

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