612 H. LASER AND LORD ROTHSCHILD I949determnined in subsidiary manometers. These, to-gether with the readings of the main manometer atthe times of sampling, enable the 02 uptake, andthe production of respiratory CO and acid to beindependently calculated for each interval betweensampling. In this way changes with time in the ratesof 02 uptake, total CO production and of R.Q. canbe obtained.

4. Two forms of the apparatus have been de-signed and are described. The theory ofthe method isdeveloped and a simplified way of computing theresults is given.

The instruments were made by Messrs W. G. Flaig andSons, Waterloo Road, London, N.W. 2.

REFERENCES

Dickens, F. & Simer, F. (1930). Biochem. J. 24, 905.Dickens, F. & Simer, F. (1931). Biochem. J. 25, 973.Dixon, M. & Keilin, D. (1933). Biochem. J. 27, 86.Laser, H. & Rothschild, Lord (1939). Proc. Roy. Soc. B, 126,

539.

Sumerson, W. H. (1939). J. biol. Chem. 131, 579.Warburg, 0. (1924). Biochem. Z. 152, 51.Warburg, 0. (1925). Biochem. Z. 164, 481.

Carotenoids, Vitamin A and 7-Dehydrosteroid in the Frog(Rana temporaria)

BY R. A. MORTON AND D. G. ROSENDepartment of Biochemi8try, Univer$ity of Liverpool

(Received 2 March 1949)

Understanding of the biochemistry of carotenoids isrudimentary except as to the part played in theanimal economy by a few of them as precursors ofvitamin A. In a wider sense the animal biochemistryis complicated by species differences. Thus someanimals absorb and retain so little of either thehydrocarbons or their hydroxylated derivatives asto suggest that they are not needed, whilst otherspecies absorb relatively large quantities of differentcarotenoids selectively and retain them distributedover various organs. For the carotenes it could beargued that they are only absorbed as such when thecapacity of enzyme systems in the gut to transformthem is exceeded, and that the pigments, of nospecial value to the body, are slowly disposed of.Such an argument, however, neglects the regular oc-currence of carotenoids in eyes, yellow bone marrowand corpora lutea and is of little value in respect tothe assimilation and metabolism of hydroxylatedcarotenoids like lutein and zeaxanthin.As a subject for detailed study the frog has several

special advantages; carotenoids (including both fi-carotene and lutein) are widely distributed in thebody; the species can be studied through the lifecycle, as embryos, as tadpoles and as immature andmature frogs; there is amarkedseasonal cycle in bothsexes and the reproductive phase entails a heavydrain on body reserves, especially in the female.Moreover, as an amphibian, the frog provides a linkbetween work on mammals and fishes. There are,therefore, numerous factors potentially capable ofcorrelation with data on the distribution of caro-

tenoids so as to lead to hypotheses concerning thefunctions of these substances.For 10-11 weeks after using up the residual yolk

the tadpole is mainly vegetarian, but when meta-morphosis is complete, frogs become mainly carni-vorous. Adult frogs build up large fat reserves inearly suxmmer, and from June to August the testes orovaries enlarge considerably. The testes then shrinkand during the next few months vary little in sizeuntil the spermatozoa become fully ripe; the ovariesgo on developing, but after spawning in March theshrunken oviducts are orange in contrast to theirearlier paleness. The corpora adiposa of Amphibiaare associated with the gonads; in the frog they varyfrom strands of orange tissue to large cream-coloured lobes. After surgical removal of one fatbody the ovary on the operated side remains smallcomparedwiththe other. Accordingto March (1937)sexually mature frogs have a body length (snout-cloaca) of about 4-8cm. and weigh about 10g.Applied with caution, body length is a guide to age.

FAT SOLUBLE SUBSTANCES IN THE FROG

Earlier work on carotnoids and vitamin A. L6nnberg(1929) reported the presence of a 'carotene-like pigment' inthe skin (Rana temporaria and R. e8culenta). Dietel (1933)noted the presence of lipochrome in skin, ovaries and liver,but thoughtit consisted wholly ofoarotene. Manunta (1934)found 'xanthophyll' and much carotene in the skin ofR. e8culenta. Rand (1935) observed vitamin A to be presentin frog and toad livers with lipochrome pigments detectablein skin, liver, ovaries and eggs, oviducts, testes, kidneys,

CAROTENOIDS AND VITAMIN A IN THE FROGlungs, spleen and fat bodies. He did not discriminatebetween free 'xanthophyll' and esterified 'xanthophylls' sothat his analyses are incomplete. He used too few frogs forhis work to be more than exploratory. Brunner & Stein(1935), using phase partition and chromatography, con-cluded that, in 1. esculenta, ,-carotene and lutein (free oresterified) were the only carotenoid pigments present. Inthe fat bodies, liver and skin, the 'xanthophyll' was all esterform, but free lutein also occurred in the ovaries. Zech-meister & Tuzson (1936), starting from 491 frog livers,prepared crystalline specimens ofliver carotenoids. The skin,ovaries, fat bodies and livers of fourteen females (R.esculenta) were quantitatively analysed by chromatographicand spectroscopic methods. Three fractions were separated:(a) epiphasic pigment, (b) free hypophasic pigment, and(c) esterified hypophasic pigment, the latter always pre-dominating; (a) consisted of cx- and ,8-carotene, the fi-isomeride preponderating; (b) was a mixture of lutein andzeaxanthin. Many unidentified zones were observed duringthe chromatography, but it is not clear to what extent thesubstances responsible were metabolic products or in vitroartefacts. Other workers include Bartz & Schmitt (1936),van Eekelen (1934), Wald (1935), Ackermann (1938),Gillam (1938) and Lederer & Rathmann (1938).

Origin of ingested carotenoid8. Relatively little isknown about the carotenoids ofthe various species ofinsects, snails and worms which form the diet offrogs, although f-carotene and lutein probably pre-ponderate. Judging from the contents of the ali-mentary tracts of frogs (in which chlorophyll isalways present) a large proportion of the ingestedcarotenoid is in the form of plant material un-absorbed from the digestive tracts of insects eaten.From the point of view of carotenoid intake the frogis indirectly vegetarian to the extent that it makesuse of plant pigments which have not previouslyentered the body proper of animals.

GENERAL PLAN OF THE PRESENT WORKNeither the design of the experiment as a whole northe interpretation of data is easy. If it had beenpossible towork on 600 frogs individuallyrather thanin batches of twenty, the plan could have beendifferent and the data obtained could have beentreated statistically. Many of the analyses, how-ever, would have been impossible on organs fromsingle frogs. The choice lay between making ageneral survey with its attendant limitations andgreatly restricting the scope of the work. Onbalance, taking into account the paucity of informa-tion in the field, it seemed better to secure theanalytical data which time permitted on samplesconvenient to deal with, than to select particularvariables for study on larger numbers of frogs.

EXPERIMENTALAnimnw8

The frogs were obtained from a commercial supplier(Norwich district) and delivered at monthly intervals. Each

batch consisted of about twenty males and twenty females.The sexeswere segregated on arrival. The frogs were weighed,killed by pithing, and dissected. The various organs, livers,kidneys, fat bodies, etc. from twenty animals ofthe same sexwere combined and weighed. Each sample was then ana-lysed for total fat and the fat in turn analysed quanti-tatively for as many of the following as seemed appropriatein each case: (a) vitamin A (free and esterified), (b) totalcarotenoids, (c) carotene, (d) 'xanthophyll', (e) free 'xantho-phyll', mono-ester and di-ester. Within a batch, the work onone sex was completed before beginning on the other.

Batches of frogs were obtained from late March toDecember in 1946 and J947 and some in 1948. The only partof the annual cycle which was not studied was the period ofactual hibernation. Ova were obtained at all stages ofmaturity and tadpoles at all stages between the fertilizedegg and the young frog. At various periods one of us(D.G.R.) collected animals from Hillside, near Southport,Lancashire, for additional tests. These included the tadpolesand young frogs (R. temporaria) and tadpoles of the natter-jack toad (Bufo calamita).

Chemical methodsOutline. Specimens of tissue were ground with sand and

anhydrous Na2SO4 and extracted with ether. The solventwas removed and the lipid made up to volume with lightpetroleum. Measured volumes were taken for the deter-mination of vitamin A, carotenoids and fat. Chromato-graphic methods were used to separate the components andthe quantitative determinations were based on data obtainedwith the Beckman photoelectric spectrophotometer. Ex-posure of extracts to light was avoided so far as possible.The extracts were not subjected to heat treatment at anystage, except in removing solvent in the fat determination.

Material for analy8i8. The frogs, which had been kept intanks, supplied with water but no food, were roughly driedwith blotting paper before weighing and dissecting. Afterweighing, the frogs were killed and dissected as quickly aspossible. Before the viscera were weighed, adhering coelomicand other fluids and clotted blood were removed; theweighings were made without delay.

Solvents. Diethyl ether was always freshly distilled overa little reduced Fe before use. Light petroleum (b.p. 40 60°)must be practically free from 'polar' impurities; the com-mercial product is variable and must either be rigorouslypurified or selected for chromatography. A consignment ofgood quality petroleum was reserved for this study. Theacetone was of ordinary reagent quality and the CHC18 ofcommercial B.P. quality. Ethanol and cyclohexane werespecially purified for spectroscopy.Totl fat. The portion of the ethereal extract which was

soluble in light petroleum was weighed after dehydrationwith ethanol and recorded as 'total fat'.

Extraction. The tissue was quickly ground, a little at atime, in a mortar with about five times its weight of an-hydrous Na2SO4 to which fine acid-washed silver sand wasadded. Tadpoles were treated similarly without dissection.The well-ground material was transferred to a beaker andcovered with ether. After standing 1-2 hr. in the dark, theyellow extract was decanted through a sintered glass filter(G4) under suction. The solid residue was then transferredto the filter cup and washed with successive portions ofether until the filtrate became colourless. The solid wasthen returned to the beaker and left to stand under ether for

613

R. A. MORTON AND D. G. ROSEN

0.5-1 hr. and ifitered. A third extraction, during which theether was boiled for a short time, yielded no more colour butremoved residual vitamin A. In some cases the finely groundtissues were allowed to stand overnight under ether in thedark in a corked flask. The solution was decanted on thefilter and the residue washed with ether until no morepigment came through. Skin, muscle, lungs, oviducts,oesophagus, stomach and intestines were extracted over-night, other tissues as quickly as possible. In all cases, theether was removed by distillation under reduced pressureat room temperature. The residue was dissolved in lightpetroleum and made up to volume. A small amount ofanhydrous Na2SO4 was added if the solution was not per-fectly clear. A measured portion of the extract was used fordirect estimation of vitamin A, the light petroleum beingremoved and replaced by a suitable volume of cydohexane.

Chromatographic separationCommercial bone meal was 'defatted' by means of boiling

acetone. The fraction which then passed through a 120British Standards Specification sieve was used for chromato-graphy. When such material is stirred with acetone, thesmallest particles do not settle readily, and if the liquid isdecanted the 'fines' can be removed. This is an advantageas otherwise acetone eluates may be cloudy. Alumina(Savory and Moore) which had been in stock in the labora-tory for some years and had become rather weakly adsor-bent proved well suited to the separation of carotenoids.Type 'O' alumina (Peter Spence and Co.) could be repro-ducibly weakened by stirring in definite proportions ofwater.A chromatographic tube of 18 mm. internal diameter was

used throughout this work. A column of bone meal 3 cm.in height sufficed for the separation of vitamin A ester fromthe free vitamin and a 4 cm. column for carotenoid separa-tion. The adsorbent was poured dry into the tube, lightpetroleum was added and the bone meal stirred with a longthin metal rod to liberate trapped air bubbles. Strongsuction was applied, but from then onwards the column wasnot allowed to run 'dry'. The initial adsorption of soluteswas effected slowly under gravity alone. Development andelution were more rapid at 120-150 drops/min. undersuction. Small volumes of eluate were aimed at and colour-less intermediate fractions obtained in carotenoid analyseswere discarded. Vitamin A esters were eluted completely by100 ml. of light petroleum and the free vitamin by means ofa little acetone. Solvents were removed under reducedpressure and the residues made up to suitable volume incyclohexane.

Carotene (in some cases contaminated with esterified'xanthophyll') was carried through the bone-meal columnby not more than 25 ml. of light petroleum. A measuredportion of this eluate was put through a similar adsorptiontube containing a 4 cm. column of alumina; here 30 ml. oflight petroleum carried down all the carotene and theesterified 'xanthophyll' was washed through with acetone(10-20 ml.). The free 'xanthophyll' retained on the bone-meal column was eluted with acetone. Throughout thispaper 'xanthophyll' refers to a fraction consisting mainlyof lutein, but possibly containing some zeaxanthin and alittle kryptoxanthin.

For a more complete separation of fractions containinglittle vitamin A a fresh portion of extract was adsorbed onalumina, the carotenes eluted by means of light petroleum,

the 'xanthophyll' di-esters by 1% of acetone in lightpetroleum (v/v) and the mono-esters by 8% of acetone inlight petroleum (v/v). In some cases the 'xanthophyll'fraction from bone meal was eluted with acetone, the solventcompletely removed and replaced by light petroleum andthe solution chromatographed on alumina. When necessary,di-esters and mono-esters can be selectively eluted frombone meal by means of 2% (v/v) and 8% (v/v) CHC13 inlight petroleum, respectively. The final fractions were madeup to volumes suitable for spectroscopic examination. Inreplacing one solvent by another heat treatment wasavoided. The visible spectra of the three 'xanthophyll'fractions are reproduced in Fig. 1. If the extracts weresaponified before chromatography, the 'xanthophyll' frac-tion was substantially homogeneous.

430 450Wavelength (m,u.)

470

Fig. 1. Visible absorption spectra in acetone of the threeforms of 'xanthophyll': (1) free xanthophyll; (2) xantho-phyll di-ester; (3) xanthophyll mono-ester.

Spectroacopic determinationsVitamin A. Vitamin A was determined by the intensity of

absorption at 328 mju. and the gross El "" values werecorrected for irrelevant absorption by the method ofMorton & Stubbs (1946). The separation of esterifiedvitamin A from the free alcohol was carried out by themethod of Glover, Goodwin & Morton (1947) using bonemeal as chromatographic adsorbent. As a rule the amount offree vitamin A was small and was best determined by thedifference between the total (corrected) and ester (corrected).

Carotenoid8. The spectra of nearly all the fractions agreedvery closely with those of the corresponding purified caro-tenoids from other sources when the criteria adopted were(a) wavelengths of maximum absorption in specified sol-vents, and (b) relative intensities of absorption at maximaand minima (Fig. 2). However, certain carotenoid fractions,from intestines and eyes, for example, exhibited irrelevantabsorption. This was particularly noticeable in the lipidsfrom frog eyes (Fig. 3). The colour is due mainly to 'xan-thophyll' and the curve may be corrected for irrelevant

614 I949

CAROTENOIDS AND VITAMIN A IN THE FROG

8

6~~~~

0~~~~0*

*Id

Z4,

2

400 440 480Wavelength (mu.)

Fig. 2. Comparison of visible absorption spectra of purecarotenoids and frog carotenoid extracts: - , luteinin ethanol; x x x x, frog xanthophyll in ethanol;- - -, ,-carotene in n-hexane; ....... frog carotene inn-hexane.

_ .--~ '\_

O 0

.0C

x

420 440 460 480Wavelength (m,u.)

Fig. 3. Visible absorption spectra of frog-eye extracts:, whole extract (light petroleum 40 60°); --,

total 'xanthophyll' (acetone); ......., corrected 'xan-thophyll'; ----- irrelevant absorption.

absorption by the general method of Morton & Stubbs(1946) on the basis of the extent of distortion of themean 'xanthophyll' curve (see Fig. 2) of extracts of otherorgans. The relative absorptions at the three wavelengths448-5, 463 and 476 mj. are E = 1, 0-724, 0.881 respectively,and E8.5 inhm. (corr.) =E1 x 2-216 -E2 x 4688 +E3 x 2472,where E1, E2 and Es are observed values at the three wave-lengths, respectively.

It was sometimes necessary to examine solutions so dilutethat the photoelectric photometer was perforce used outsidethe recommended range of extinctions. The instrument wastherefore calibrated over the range E =0.01 to E= 1 0 usingcarotene in light petroleum and 'xanthophyll' esters inacetone or light petroleum. When readings below E= 02were obtained with frog extracts, corrections derived fromthe calibration curves were applied. In the cases of mono-esterified and free 'xanthophyll' fractions in acetone similarcalibration demonstrated that no such correction wasnecessary. The data in Table 1 were used as a basis forcalculations.

Table 1. Spectrophotometric data on carotenoid8,u8ed in the analytical work

Substance,-Carotene

LuteinVitamin AFrog 'xanthophyll'Frog 'xanthophyll'di-esters

Frog 'xanthophyll'mono-esters

(mg.) EI C Solvent449*5 2550 Light

petroleum446-5 2550 Ethanol328 1700 CycloHexane448-5 2640 Acetone448 2550 Acetone

448 2600 Acetone

Remarks(a)

(b)(a)(c)(c)

(c)

(a) Determinations in this laboratory; (b) Zscheile,White, Beadle & Roach (1942); (c) values determined in thecourse of the present work relative to El C. 2550 for purefree lutein.

If, in determining the 'total carotenoid' per frog in agiven type of tissue, x frogs were used to provide an extractin y ml. and the solution had to be diluted by a factor z so asto bring Em,,a. for a 1 cm. layer to a convenient figure; then

Ez EyzQlOOx/y lOOx

The use of Q avoids the difficulty that 'total carotenoid' isa mixture of components in which neither A... nor E 1 %m isnecessarily exactly the same. Since, however, E1 at thevisible maximum is of the order 2500, i.e. E is about 2*5 fora 1 cm. layer of solution of concentration 1 mg./100 ml.,Eyz/250x is the weight of solute per frog in mg., so thatroughly 0-4Q=mg. total carotenoid/frog.

In this investigation the number of analyses was neces-sarily very large and the preparation of material time-consuming. In choosing methods the aim was the greatestaccuracy compatible with speed. It was found to beessential to complete the tests on livers, kidneys and eyes,so far as vitamin A was concerned, in one working day;standing overnight might have caused a loss of as much as40% of the vitamin. The intact organs could, however, bekept at 0° without appreciable loss (Table 2). The method ofdecomposing the tissue by boiling with ethanolic KOH andtesting the non-saponifiable extract was tried side by side

Vol. 45 615

R. A. MORTON AND D. G. ROSENwith the procedure already described, and was found to beattended by significant losses or isomerization of caro-tenoids (Table 3).

Table 2. Stability of carotenoids infrog tissuesstored at 00 after disection

Period of Totalstorage carotenoid

Tissue (days) El% *

Liver 0 0-09761 0-09983 0-0968

Kidney 0 Not tested1 Not tested

Muscle 0 0-004873 0-00482

Totalvitamin A

El % (corr.)*0-1170.1150-11305430-557NilNil

TissueCock liverHorse liverHare liverFrog skinFrog liverFrog liverFrog testes

Total carotenoidE1 cm.

(a) (b)0-008 0-00720-0192 0-0179

Negligibly small0-031 0.0300-080 0-0690-227 0'1750.053 0-046

Vitamin AE1 ' (corr.)*

(a) (b)Not measured0-248 0-2630-034 0-035

Negligibly smallOl05 0-1070-278 0-243

Negligibly small

* Calculated by the procedure ofMorton & Stubbs (1946).

Samples of frog carotenoid fractions with accompanyinglipids were used to ascertain the magnitude of manipulativelosses in the chromatographic procedures. Over the relevantrange, recoveries of 97-99% were obtained. Similar testswith free and combined vitamin A provided equally goodrecoveries.The magnitude of the correction for irrelevant absorption

at 328 m,u. in tests on liver extracts is illustrated in Fig. 4,and on extracts from eyes and kidneys in Fig. 5. The fulllines in Fig. 4 show irrelevant absorption at its greatest inthe present series (in all there were three such cases); thebroken-line curves illustrate the normal case in which theirrelevant contribution is about 20% (thirty cases). Thecarotenoid contribution amounted at 328 m,u. to only 3-4%of the total absorption. The need for correction at 328 mg.is much more obvious in the extracts from eyes and kidneys,but the validity ofcorrected estimates for vitamin A contentwas confirmed using the SbCla colour test.When the ultraviolet absorption of an extract suggested

the absence of vitamin A, the colour test was used toprovide confirmation.

RESULTS AND DISCUSSION

The experimental findings are recorded in tablesand figures.

Lipochrome8 and vitamin A in tadpolemand young frog8

Pre8ence of chlorophyll. The absorption spectraof chromatographic fractions containing 'xantho-phylls' show some chlorophyll to be present also.

0-8

280 '320Wavelength (mpu.)

360

Fig. 4. Ultraviolet absorption spectra of liver extractsillustrating the magnitude of the correction:typical spectrum; , atypical spectrum. In each setthe top curve is observed, the middle curve corrected andthe lowest curve represents irrelevant absorption.

0.6

0

x

.I.0

300 350Wavelength (niu.)

Fig. 5. Typical ultraviolet absorption spectra of eye(- ---) and kidney ( ) extracts, showing gross,corrected and irrelevant absorption.

* Calculated on wet wt. of tissue.

Table 3. Compariaon of analyse8 for carotenoids andvitamin A carried out on (a) ether-extractable lipidand (b) non-8aponiflable ether extract after treatingtissue with ethanolic KOH

616 I949

I

CAROTENOIDS AND VITAMIN A IN THE FROG6Thus, tadpole extracts exhibit maxima at 662-664, 615 and 432-434 m.; E662mpL/E615m. = 4-2.Harris & Zscheile (1943) give for chlorophyll-aAm.. 661-5, 615-5 and 431-5 mjA; E661i5ml/E615.5 mIp.= 6-3. The low value for the ratio of theintensities of the two chlorophyll bands (Figs. 6and 7) suggests the presence of chlorophyll-b. Byapplying the method ofComar & Zscheile (1942) to a

Wavelength (m,u.)

Fig. 6. Visible absorption spectra of frog tadpole 'xantho-phyll' in acetone: , 'xanthophyll' from chromato-gram; -----, computed chlorophyll contribution;-----, xanthophyll contribution.

'xanthophyll' contribution was obtained by differ-ence. This substance, which showed a maximum at412 mgi., was probably a degradation product ofchlorophyll; it was easily removed by adsorption onalumina and the anomalous 'xanthophyll' absorp-tion was corrected by subtraction (Fig. 7). Caro-tenoids are decomposed by exposure to light in thepresence of chlorophyll and oxygen (Pepkowitz,1944); it is necessary, therefore, to protect the

.0

v

._1

._

400 450 500Wavelength (muL.)

650

Fig. 7. Visible absorption spectra of natterjack tadpole'xanthophyll' in acetone: , 'xanthophyll' fromchromatogram; - - - -, computed chlorophyll contri-bution; -----, absorption of 'xanthophyll' plus arte-fact; x - x - x, artefact contribution; ......., 'xantho-phyll' contribution.

fresh ether extract of tadpoles, chlorophyll-a was extracts as much as possible. Vitamin A in traces isfound to preponderate over chlorophyll-b by a factor very difficult to detect in such mixtures, and the onlyof 7-5 to 1. The observed absorption in the region way in which its presence was demonstrated in400-500 m,u. is therefore a summation of contribu- natterjack tadpoles was by the antimony trichloridetions from 'xanthophyll' and 'chlorophyll'. The colour test applied to extracts from the dissectedabsorption in the region 600-700 m,u is due solely to eyes.the latter, and from the absorption curve for pure Occurrence of carotenoids. The number of tadpoleschlorophyll-a, the curve from the red region can be usedwas ample forthemethods applied, andthe datacontinued through to the violet using the following obtained are shown in Tables 4-6. The tadpolesrelative E values: were grouped in accordance with the stages of de-A 662 400 412 420 430 velopment A-Hwhich can be fitted into an approxi-El 0-976 0-7 0-913 0-875 1-218 mate time scale (see Fig. 8).

A 432 440 450 460m,uComment. The chlorophyll is almost certainly all

E 14225 04595 0 074 0-018 present in the digestive tract. The tadpoles werekept for 3 days in captivity after collection, butThe 'xanthophyll' contribution in tadpole extracts although they were active and faecal excretionis then obtained by difference (Fig. 6). In two copious, it was found that those collected at stage Asamples, the 'chlorophyll' and 'xanthophyll' were had to be kept for 9 days before chlorophyll dis-separated by chromatography on weakened alumina appeared. In tadpoles caught at different ages theand the validity of the difference method confirmed. chlorophyll content is considerable in stages B, C

In extracts of natterjack tadpoles the chlorophyll and D, less in E, very low in F and negligible in G.contribution was computed in a similar manner, but This suggests a decrease in foodintake which is bornean additional maximum near 420 m,. due to the out by the weights and the carotenoid analysespresence of an artefact was observed when the (TabIe 4).

Vol. 45 617

R. A. MORTON AND D. G. ROSEN

Table 4. Carotenoids in tadpoles of Rana temporaria at various stages of development

(Chlorophyll was present in A-F, not detected in G and H.)

StageA, young tadpoles, 10-20 mm.B, tadpoles, 20-30 mm.C, tadpoles, 30-40 mm., rearleg just seenD, rear legs less than 4 mm.E, rear legs greater than 4 mm.F, fore limbs formed, some freeG, all four limbs well formed,long tails persistingH, small frogs, tails almostresorbed

Date andnumber used(in brackets)2. v. 47 (312)2. v. 47 (236)20. v. 47 (75)

4. vi. 47 (82)4. vi. 47 (52)4. vi. 47 (73)

10. vi. 47 (88)

10. vi. 47 (93)

Table 5. Rate of disappearance of carotene and'xanthophyll' during metamorphosis of tadpoles

Average amounts of pigments(1tg./tadpole)

, ~~~AStages Stages

C, D and E* F, G and HtCarotene 0-33 0-25'Xanthophyll' 131 0-80

* Feeding freely. t Fasting.

Loss(%)2438

Table 6. Rate of disappearance of carotene and'xanthophyll 'from young tadpoles caught at stage Aand kept in captivity withoutfood

Pigments expressed as(.ug./tadpole)

Carotene' xanthophyll'

1 ~~~ATested 3 days Testecafter capture after c

0-0116 0 (0-112 0@(

* Chlorophyll absent.

- Lossd 9 days (%)capture* (approx.)0093 20087 22

Average wt.(g.)0 0490-120-32

0-220*250-220-19

0.15

Carotene/tadpole(pg.)0-0120 0330*31

0-270*400-250-23

0-25

'Xantho- Ratiophyll'/ 'xantho-tadpole phyll'/

(.pg.) carotene0*11 9*70*30 9.11*78 5-6

0-93 3-41-21 300-92 3-70*72 3*1

0*76 2-8

'utilization' as measured by rate of disappearance.If, during the period of metamorphosis, carotenehad been used for the synthesis of vitamin A, thehydrocarbon might have been expected to disappearmore quickly than the 'xanthophyll' (mainlylutein in the tadpole). There is no doubt thatvitinA is formed in the tadpole. Natterjack tadpoleseach contained about 0*027 ,ug. in the eyes and newlymetamorphosed frogs about 0 1 ug.

The amount of carotenoid in the original eggs isless than one quarter of that present in active tad-poles. Now the ratio of 'xanthophylls' to carotenesin the plant world, although variable, is only veryrarely outside the range 3-10. In young tadpoles theratio is initially about 9 and falls to about 3 at thetime of metamorphosis and then falls further inyoung frogs. Up to maximum growth 'xanthophyll'storage increases more rapidly than carotenestorage. The abrupt fall in 'xanthophyll' level whenthe rear legs first appeared obviously needs con-

firmation, but the very small concomitant drop incarotene affords a hint that it is not illusory. The factthat little or no food is eaten during metamorphosisallows the disappearance of carotenoids to befollowed. The data given in Fig. 8 and Tables 5 and 6suggest that the main process does not discriminatesharply between carotene and 'xanthophyll'. Thechange in the 'xanthophyll '/carotene ratio recordedin Table 4 is not then simply a matter of selective

May June JulyTime

Fig. 8. Carotenoids in the developing tadpole: (a) totalcarotenoid; (b) total 'xanthophyll'; (c) carotene. Thestages A-H are specified in Table 4.

It is now clear that the main, ifnot the sole site ofconversion of carotene to vitamin A in mammals isthe absorptive portion of the gut, and it seems veryprobable (although obviously difficult to prove) thatthe same holds true for the tadpole. It is very un-likely that the 'xanthophyll'/carotene ratio in thekind of vegetation ingested is ever as high as 9; theappearance of this ratio in the early stages of de-velopment (A and B) suggests a selective utilizationof carotene in the gut wall. On this hypothesis, asdevelopment proceeds and food intake increases, the' carotenase' enzyme system becomes unable to copewith more than a fraction of the carotene presentedto it. The residue will follow the normal pathway oflipid absorption, and the 'xanthophyll '/carotene

618 I949

CAROTENOIDS AND VITAMIN A IN THE FROG

ratio will fall until it reaches a figure much nearer tothat of the diet. The carnivorous young frogs willcontinue to convert a proportion of ingested caro-tene and to absorb carotene and 'xanthophyll' non-selectively.

It is possible that in the future the hatching offrog spawn and the growth oftadpoles in captivity onartificial diets may be of service if either carotene or'xanthophyll', or both, can be excluded.

Lipochromes and vitamin A in adult frogsVitamin A. There does not appear to be any pre-

formed vitamin A in the diet ofthe frog. The vitaminformed in vivo from provitamin is found in the liver(about 85 %) and in the eyes and kidneys in approxi-mately equal amounts (Tables 9 and 11). In eachsite it occurs mainly as ester. The amount of vitaminA in kidney (about 30 ,ug./g.) is verymuch larger thanthat found in mammalian kidneys.

Carotenoid distribution. Carotene as such is lessefficiently absorbed than the hydroxylated caro-tenoids since the 'xanthophyll'/carotene ratios areapprox. 3: 1 in the diet, 2: 1 in the faeces and, in thebody, 4 7:1 for females and 5-7: 1 for males (October1947).

In contrast with the restricted distribution ofvitamin A, carotenoids occur in all the organs studied(Table 9). They also appear to occur in nerve tissueand in glands such as the thymus. If the amounts ofcarotenoids at different sites are expressed in termsof that present in the skin (chosen for comparisonbecause of its relative constancy), the results inTable 7 give an idea of the approximate distributionover the body. In males, the skin, liver and muscleare important; in the female the gonads dominatethe situation (Table 7).

Table 7. Relative amounts of total carotenoidin frog organs

(These are mostly seasonal averages, without distinctionbetween the sexes.)

Skin*Ovaries and mature eggsLiverMuscle (total leg)tOviductsImmature ovariesStomachTestesSmall intestine, fat bodies, kidneysOesophagusTonguePancreas, large intestine, lungs, eyes,heartSeminal vesicles, spleen

1

2-650-70-170-120-10050 0350-23-0-280160*090-004-0-008

Traces* Skin carotenoid is taken as unity, because the absolute

amount is large and is less influenced by sex and seasonthan other tissues rich in carotenoids.

f Carotenoids of leg muscle/ventral and side muscle/armmuscle =36/2/1.

Table 8. Weights offrog organs expressedas percentage of body weight

OrganTestesOvariesOviductsLivers (male;Fat bodies(male)

SkinLeg muscleKidneysPancreasStomachLungs (male)TongueEyesHeart

Minimum0-320-925 (Apr.)1-47 (Apr.)114 (Apr.)) 1-3 (Apr.)

INearly 0 (Apr.)(Nearly 0 (Apr.)Approx. 10Approx. 15

0 3-0 50*06-0-13

Approx. 1-0I0-42)0-250-8

Approx. 1-0Approx. 0-25

Maximum3-66 (Aug.)

36-0 (Mar.)17-5 (Dec.)3-6 (Nov.-Dec.)2-3 (Oct.-Dec.)8 (Aug.-Sept.)7 (Aug.-Sept.)

Although the left fat body is larger than the right one,the carotenoid content per unit weight is the same. Theadrenals are somewhat richer in carotenoids than thekidneys. In all tissues x-carotene seems to be practicallyabsent and the 'xanthophyll' is nearly all lutein. Verysmall amounts of other carotenoids were sometimes found,but there was too little for characterization. A few toads(Bufo vulgari8 and B. calamita) have been studied in thecourse of the present work and no significant differenceswere observed from experience on frogs.

One of the implications of the distribution data isthat the carotenoids are not merely distributed withthe fat; thus for females in October 1947 the con-centrations of carotenoids in fat varied from about0-01 mg./g. for fat bodies to 8-4 mg./g. for skin, withliver 2-7 mg./g. and ovaries 1-1 mg./g. (Table 15).The external appearance of frogs and toads oweslittle or nothing to the lipochromes present in theskin. The outer layers are rich in melanins and inpigments soluble in dilute ammonia.

Lederer & Rathmann (1938) found small pro-portions of vitamin A2 in R. esculenta liver fat, but inthe present work with R. temporaria no A2 was ob-served, confirming the finding of Gillam (1938).

Ultraviolet absorption spectra of lipidfractionsfromfrog organs

The absorption spectra of liver extracts have already beenreferred to (Fig. 4). Observations on extracts from otherorgans are recorded. Selective absorption of undeterminedorigin in the region 280-300 mju. was recorded in lipids fromthe eyes of frogs and toad (Fig. 5) and also rather erraticallyin frog kidneys. With one exception (November 1946) thetestes extracts showed continuous end-absorption over therange 250-400 mit. The aberrant sample showed an inflexionnear 265 mp.

Extracts from ovaries were more interesting.Material from the mature ovaries of frogs in the pre-hibernation period exhibited the well-resolved ab-sorption shown in Fig. 9. The characteristic curve

VoI. 45 619

R. A. MORTON AND D. G. ROSEN

Table 9. Di8tribution offat, carotenoid and vitamin A in the organs of sixty male frogs,divided into three main groups

Mean body length Mean weightGroup (Om.) (g.)A 6-6 22-8B 5.6 14-2C 5.1 11.0

In table below means were not measured; a dash (-) means 'not measured'.Wt. as % Wt. of fat Total carotenoidbody wt. (mg./frog) Q x 102*

0-0580*0610 030

0-1170-1050-120

0-350-350-35

1-201-241*23

5-65.42-5

0-330-080-02

Carotene(,Ug./frog)

0-158

'Xanthophyll'(g./frog)

1-40

0-110-060057

0-290-2350-18

4-12-71-7

0-330-220-17

0.590-380-32

0-770-560-40

0-861-021-30

2-312-311-92

0-630-740-72

1-091-121-27

11 611.111.1

17-315B614-5

3-62*92-2

4-463*842-32

3-43-31-25

13-011-57-2

0-110-0820-078

0-480440-34

16-212-69.7

18-310-66-8

12-58-56-9

3-422-041-54

* See p. 615 of text.

with maxima at 292-5, 281-5, 271 and 261m. was

reproduced in the non-saponifiable fraction. Thespectrum is qualitatively indistinguishable fromthat shown by provitamins D, e.g. 7-dehydro-cholesterol and ergosterol. Without using a largeamount of material it is not possible to decide whichof the several substances known to exhibit thisspectrum occurs in the frog ovaries. Applying a

0-520-490-35

7-255-022-19

1-040-970.69

1-241-160.90

43-627-824-4

10-66-95.0

Vitamin A(.sg./frog)

000

000

5.32-41-6

0

0

0

3.02-32-1

39-620-612-1

0

0

0

0

0

0

0

0

0

0

0

0

correction procedure based on the method ofMorton & Stubbs (1946) using reference data ob-tained on highly purified ergosterol (which isspectroscopically almost identical with 7-dehydro-cholesterol) the corrected E",. 281-5 m,. value was0*117, corresponding with a concentration of 1 in2430 in the ovaries [El o, (ergosterol) = 285]. A cal-culation based on the work of Boyd (1938) indicates

OrganFat bodies

ABC

PancreasABC

KidneysABC

TestesABC

EyesABC

LiverABC

TongueABC

StomachABC

SkinABC

Leg muscleABC

620 I949

CAROTENOIDS AND VITAMIN A IN THE FROGthat mature ovaries contain about 0-8 % of chole-sterol. It thus appears that the ovarian steroidscontain approx. 0-5% of 7-dehydrosteroid.

Cc0._iua.4x C

co

250 275 300Wavelength (m,u.).

325

Fig. 9. Ultraviolet absorption spectra of the extract ofmature frog ova in ethanol: (a) gross absorption;(b) corrected curve; (c) irrelevant absorption.

c 04.2

x

U'

260 280Wavelength (m,u.)

300

Fig. 10. Ultraviolet absorption spectra offrog ova extracts:A, mature ova just prior to oviposition (ethanol);B, Decemberovaimmaturethrough deprivation (ethanol);C, immature ova from frogs which yielded mature ova

(curve A) (in ethanol); and D, immature ova from springfrogs 1947 (cyclohexane).

The characteristic spectrum only appears whenthe ovaries are mature, but it persists through theperiod of hiberation and is seen (Fig. IOA) justbefore oviposition occurs. There is some loss of defi-nition and possibly a decrease in intensity, but it isdifficult to say how much of the apparent loss is

fictitious, since the protein layer and water are

accumulated at this time. Curve C (Fig. 10) showsthe absorption spectrum of an extract of inunatureovaries obtained from the same frogs as the ripe ovain March 1948. Curve D refers to immature ovariesobtained in the spring of 1947 and B to ovariesfrom frogs which had fasted for a long time(p. 625). The weak selective absorption at 330 m,u.(Fig. 9) is not due to vitamnin A or carotenoids.

The data on extracts from other organs show no well-defined spectra, but there are numerous indications of thepresence of traces of absorbing constituents. Much laxgeramounts of material would have to be used in following upthese observations.

The occurrence of the ergosterol-like bands inmature ovaries is surprising. It suggests that thehypothesis ofa role for 7-dehydrosteroids other thanthat of a provitamin D is worthy of study.

Seasonal variation8 in carotenoids and vitamin A

A considerable body of metrical data accumulated duringthe study of some 600 frogs, the main features of which aresummarized in Tables 8 and 10. The quantitative picturewhich emerges from a study extending over only twoseasons is in some respects rather tentative, but it is likely tobe broadly correct. In 1946 the liver fat for both sexesshowed a 'xanthophyll'/carotene ratio (X/C) of 1 in theautumn, but a year later X/C was about 3 (Table 12). The

Table 10. Size and weight offrogs used atdifferent dates in the present work

(The average body lengths were computed from the dataof March (1937), except those marked with an asterisk.)

Date ofdissection14. viii. 4619. viii. 469. ix. 46

11. ix. 4617. x. 4621. x. 466. xi. 46

12. xi. 469. xii. 46

16. xii. 461. iv. 477. iv. 47

13. iv. 4712. v.4728. v.478. vii. 47

22. vii. 473. x. 478. x. 47

30. x. 4730. x. 4730. x. 47

Number

Males Females

- 181820_ 1918 -

1820_ 2019

2113

18 -

1420

1914

1917

17202020

Averagebody wt.

(g.)27-520-430-525-626-826-729-334.525-431-129-522-714-520-316-325-319-913.116-822-814-211-0

Averagebody length

(cm.)6-96-57-36-96-96-57-16-96-76-76-66-26-56-56-37-16-75.5*

5.9*6.6*5.6*

5.1*

weather remained mild until early December in 1946 and thefrog fat bodies were much larger in October and Novemberthan a year later. Apart from complications arising from thefact that no two years have the same weather conditions,

)-2~~~~~~~.

/ (a

6 N-N. b.\

.4 _4 '4' 4.

4' , '4 % '

Vol. 45 621

0-i

0.

R. A. MORTON AND D. G. ROSENthe age (size) variable must be distinguished from thevariable of an annual cycle in the frog at all ages afterpuberty.A group of sixty male frogs, captured near Norwich in

October 1947, was divided into three groups. Table 9summarizes the experimental data. The weights of organs

expressed as percentage of body weight are reasonably con-

stant apart from the cases of fat bodies and eyes. No simpleexpression can be found to cover the lipochrome or vitaminA variations, but both increase with increasing age.

An experiment with a further group of sixty male frogscaught in the spring of 1948 at Hillside showed that twogroups of twenty not differing in average size (6-7 cm.)differed little in respect of skin carotenoid, liver carotenoidand liver vitamin A. A third group of the same size showedvery much less liver vitamin A and slightly less liver caro-

tenoid. The frogs in the third group, uinlike the others, were

spent (seminal vesicles empty, and testes shrunken), and, as

will be seen, it is plausible to connect that fact with thedecrease. Only rather gross signs of seasonal variations,reinforced by data on the average body length of the frogsemployed (see Table 10), can therefore be regarded as

significant at this stage in the problem.The variations in carotenoid and vitamin A content of

certain organs during the annual cycle are illustrated inFigs. 11-15. The results obtained on the first sixty malefrogs in groups A, B, C (p. 620) have been taken intoaccount in the discussion.

Vitamin A(i) Liver. Males and females showed a summer maximum

with a fall in early autumn and a rise in late autulmn(Fig. 11). There may have been a real decrease prior to

L-b0 400

bo

.' 30E

20

Aug. 1946 Jan. 1947Date of examination

Aug. 1947

Fig. 11. Seasonal variation in frog liver vitamin A:x-x-x, male; -*-v-, female.

hibernation, but there was no sign of a drop during winter.Some females shed their eggs in. the laboratory withoutpairing (April 1947) and showed low liver levels afterwards.Similarly, in 1948, the three sets ofmales already referred toshowed respectively 36-1, 36-2 and 20-77,g./liver, the lowvalue being obtained in the 'spent' animals.

(ii) Eyes. Both sexes showed a late autumn maximum anda fall in vitaminA before hibernation. Males but not femalesappeared to exhibit a winter minimum (Fig. 12).

I949(iii) Kidney. Beyond the fact that kidney storage of

vitamin A seems to be maximal in autumn, there is too littleevidence for postulating a true annual cycle (Fig. 12). Thereis little evidence that hibernating frogs deplete their vitaminA reserves, but reproduction causes a heavy drain. Thetemperature coefficient of the enzyme system responsiblefor the conversion of provitamin A to vitamin A is probablymore responsible for the summer rise in the liver reserve

than the copious feeding.

4

31-1

W_ow0

0

C-21-

.E_

11

Jan. Apr.1947 1947

Nov.1947

Fig. 12. Seasonal variation in frog eye and frog kidneyvitamin A: x, eye (male); 0, kidney (male); A, eye(female); *, kidney (female).

Table 11 shows for frogs at different seasons the vitamin Acontents of the three organs expressed as percentage of thetotal reserves. The seasonal variations suggest sex differ-ences, and show clearly that the liver is always the mainstorage organ. So far as the data go, 75-88% ofthe vitaminA occurs as ester in all sites with 80% as a typical figure.A few discrepant observations were, however, made.

Table 11. Seasonal variations in the distributionof vitamin A infrogs of both sexes

Averagebodylength

Season Sex (cm.)Spring M. 6-2

F. 6-5

Vitamin (% of total)

Liver87-088-4

Eyes Kidneys6-9 6-17-2 4-5

Suimmer M. 7-1 88-8 4-1 7.1F. 6-7 90-2 5.5 4-3

Autumn M.F.

5.5 77-5 11F7 10.96-0 82-7 9.9 7.5

Total carotenoid(i) Liver. No reduction was manifested during hiberna-

tion for either sex, in fact there may have been a small risefor males. In both males and females the decrease on matingwas followed by a rise to a summer maximum. There was

622

A

Xe

I I-I

CAROTENOIDS AND VITAMIN A IN THE FROG

then a gradual decrease continuing until December withnothing analogous to the autumnal rise in hepatic vitamin A(Fig. 13).

0-1

0@1>

*- 0.1"bO

0

.5

0*0'(

10-0

0.0

oo4

July1946

Jan.1947

Date of examination

Nov.1947

Fig. 13. Seasonal variation in frog liver total carotenoid:x - x, male; *-0, female. (Q, see p. 615.)

c 0 004

o ooo;bO

co

A 0.00!

c 0 0020

Z- o-oo:

r_ 0 001e

June1946

Jan.1947

Date of examination

Nov.1947

Fig. 14. Seasonal variations in testes and fat body totalcarotenoid: x - x, testes; 0-0, female fat body; *-U,male fat body. (Q, see p. 615.)

0'

0

c0

0

0a>

July Apr. Jan.1946 1947 1948

Fig. 15. Seasonal variation in ovarian and oviduct totalcarotenoid: x - x, ovaries; 0-0, oviducts. The ringedcross represents the total carotenoid content of mature

ova just prior to oviposition. (Q, see p. 615.)

(ii) Sex organs (Figs. 14 and 15). The gonads and also thefat bodies showed marked seasonal variations in carotenoidcontent. The testes were very poor in lipochrome aftermating and remained so until August when a sharp riseoccurred; there was some fluctuation from September to

November, but no clear-cut secondary maximum wasrecorded. Transfer to the testes seems to occur during or justafter hibernation. Immature ovaries contain very littlecarotenoid, but just prior to hibernation a thirtyfold risehad been attained. Whether or not there is a drain on ovariancarotenoids during the winter sleep cannot be stated, ascontradictory results were obtained in the 1947 and 1948seasons, but it could not be large. The oviducts showed a risein late autumn, but there was no drop in winter (when theducts are functioning actively). On spawning, the ductsatrophy and there is a sudden decrease to a low level whichis maintained through the summer.

Male fat bodies showed a marked rise in November,whereas in females the levels were lower and fairly constantthroughout the autumn. Hibernal losses occurred in bothsexes and for females especially, marked depletion occurredon spawning, with recovery during summer feeding to lowerlevels for females than for males.

(iii) Kidneys, skin and organs of the digestive tract. Nosignificant seasonal variations in kidney or skin carotenoidscould be observed for either sex. Some variations occurredin the digestive tract. Males showed summer and autumnpeaks in the stomach, but females only showed the autumnrise. In the small intestine there was a maximum in autumnfor both sexes. During winter the intestinal carotenoidswere maintained (males) or even rose a little (females).

(iv) Leg musce. A temporary rise appeared to occur in

the autumn.(v) Tongue, lungs, pancreas, eyes. There was evidence of

an autumn maximum, but the levels were fairly steady on

the whole. In the male pancreas an increase is suggestedduring or immediately after hibernation, but in the femalethere is a fall on egg-laying. In the male eye the total'xanthophyll' drops in early summer and then rises, butthere is no clear-cut autumn maximum in the female.Carotene was found in traces (<0.05 pg./eye) but 'xantho-phyll' preponderates in the eye.

Comment on seasonal variations

The foregoing results are not easy to interpretfully. A tendency for carotenoid content to riseafter heavy feeding is quite definite, but some

organs respond quickly and others much more

slowly. Furthermore, theremaybe aseasonalchangein storage sites. For example, during the earlysummer-feeding flush, the liver lipochrome in-creases markedly, yet the muscle content does notrise appreciably; during the autumn feeding this isreversed. However, it is not possible to distinguishbetween carotenoids carried with fat in lipid transferand carotenoids mobilized in accordance with thecycle of specific utilization if such exists.The gonadal cycle in the female results in a larger

storage of carotenoids in the developing ovaries thanin the whole ofthe rest ofthe body. Expulsion ofovais by far the largest cause ofcarotenoid loss, probablylarger even than the total oxidative degradation.The cycle in the testes and oviducts is not responsiveto dietary changes, but this is no more than a hint atfunctional significance. The sex difference in caro-

tenoid storage in the fat bodies recalls the dissimilargonadal sequence, for spermatogenesis is spread over

4 X

0

4 ---

7

.t-, p'6o

VoI. 45 623

624 R. A. MORTON AND D. G. ROSEN I949no more than six weeks, whereas ovarian develop- appears to do in female livers (August-Decemberment proceeds steadily until the autumn accelera- 1946), transfer of 'xanthophyll'from liver to gonadstion of growth. is the probable explanation, because the quanti-Although muscle and kidney stores fluctuate rela- tative demand of female gonads for 'xanthophyll' is

tively little it is noticeable that the absolute amount great. During spermatogenesis (when the total caro-stored in muscle (especially for males) is quite appre- tenoid is maxiimal) theX/Cratio for testes is doubled,ciable. Muscle fat and carotenoid is probably but the reason is not clear.mobilized readily. It is interesting that, althoughfrogs feed soon after mating, carotenoid storage does Free and combined 'xanthophyl'not occur until late May. There is a similar lag in liver Seasonal effect8. Table 13 shows the distributionglycogen and blood sugar increment (Smith, 1947). of free 'xanthophyll', and mono- and di-esterified

' xanthophyll'as a percentage ofthe total. AlthoughThe 'xanthophyfl '/caFrotene ratio (x/C) high accuracy cannot be claimed for the figures it is

The relative amounts of 'xanthophyll' and caro- obvious that the organs fall into two classes in whichtene stored differ markedly in the various organs. the 'xanthophyll' is respectively mainly free andFor frogs of different sizes the quotient varies as mainly combined. The significance of the data infollows: testes 1'2-1-5, liver 3-5-5-8 and stomach Table 13 is not clear, because it implies a distribution2-4-2-55, skin 55--11, muscle 7-10. There is, how- of esterases for which no explanation can be offered.ever, no uniform trend with increasing age in differ-ent organs. The data on seasonal changes are sum- (omparion offat and carotenoid storagemarized in Table 12. It is quite possible that the Since lipid is the normal vehicle for carotenoids itpicture is distorted by some local or other factor is important to knowwhether the storage and mobili-connected with the availability ofparticular kinds of zation of lipochrome is correlated with that of fat.food; in fact marked annual differences have been Tables 14 and 15 show that it is not. (The ratio fat/observed. Some of the grosser effects can, however, carotenoid (C/F) is approximate, since the figuresbe interpreted. When carotene predominates as it for total carotenoid are obtained by the roughly

Table 12. Sea8onal change8 in the 'xanthophyIl '/ carotene ratio infrog organe1946 1947

r A A

Organs Sex July Aug. Sept. Oct. Nov. Dec. Apr. Apr. May July Oct.Testes - 2.5 (?) 1.95 1.06 0-87 1-06 0-85 0-88 1-92 1-42Ovaries 7-2 6-3 7-2 7.2 6-7 7.4 7.7 11.6 9-6 112 8.2Fat bodies M. 7.7 5.7 5.0 4.9 6.7 - 16.0 10-9 7.9

F. - 5*0 4.4 - - 7-2 5-1 9-2 -Skin M. - - 5 0 4-6 5.3 4'8

F. - - 4.1 2.6 4.3 4-1 4-6 2.8Liver M. 1.12 0-72 1-97 1*59 1.19 1.46 151 - 1-57 174 3.0

F. 1.02 0.51 0-78 0-88 0-71 0.95 0.81 158 1-38 174 2.8Kidney M. - - 3-6 50 4.5

F. - - 2-9 2-7 9.3 2.2Leg muscle M. - - - - 10 7.5

F. - - 8.3 8-7Lungs M. - - - 3 0 - - -

F. - - - 3.9 9.0

Pancreas M. - - 6-8 - - 158 7.7F. - 6.2

Stomach M. - 2-1 1-7 1.8 - 2.4 - 2.1F. - - - 2.4

Intestine M. - - - 4-1 2-6 - - - 40F. - - 2.6 4-2 3.1

Large intestine M. - - - 1.9 - 0 9F. - 2-3 1-0

Tongue M. - - - - - 3-8 1.1F. - - - 1.8 2.2

Oesophagus M. - - - -- 2.4F. 4.9

Oviducts* - - - - - 0-83 0.67 0.76 0.72 1.0* Oviducts aJone fail to show 'xanthophyll' predominating at any season of the year. X/C ratio was 0-88 in Jan. 1948.

CAROTENOIDS AND VITAMIN A IN THE FROG

Table 13. Sea8onal diatribution offree 'xanthophyll' and xanthophyll mono- and di-e8ter8in frog organ8

Percentage of total 'xanthophyll' as

SeasonSummerSummerAutumnSpringSummerAutumnSpringAutumnAutumnAutumnSpringAutumnWinterWinterWinter

Sex Di-esterF. 68M. 61M. 68M. 74

52- 60

58M. 9M. 12F. 8F. 10- 11

7M. 11M. 6

Table 14. Total carotenoid8 and total fat of organs of male frogs of different Sizes

(Mean body lengths: Group A, 6*6; B. 5-6; C, 5-1 cm. Total carotenoid calculated on basis of mean El /° 450 m,. 2500.)

A

Fat Carotenoid(mg.) (pg.)4-1 1-33-6 18

16X2 5018*3 13-7

C/F(mg./g.)0-35-03-10 75

Fat(mg.)2-72-9

12-610-6

Carotenoid(.ig.)0-87

15*4348-2

Table 15. Seasonal variations (1947) in ratio oftotal carotenoid/fat infrog organs

(Ratios expressed as mg. total carotenoid/g. fat.)

OrganTestesOvariesFat bodies

Kidneys

Liver

Leg muscle

Skin

Oviducts

Sex

M.F.F.M.F.M.M.F.M.F.

April

3-4

3-33-2

May0-631-30-490-020-660-283-61*6

July0*441-120.010-010-30471-62-7

- 00342-8 3.5 313-4 2-3 8-40-3 0-3 0-2

October0-420-680*090-121.51-43-61.11-81-23-24*00-4

correct assumption of an average E1 %m 450 m,. of2500 for the constituent pigments.) Variations withsize are obviously less important than inter-organvariations. Seasonal variations (Table 15) are

striking, but again there is no correlation between fattransfer (or metabolism) and the fate of carotenoids.

Vitamin A fed to frogsOne male and one female were each given 1 drop (approx.

3500 i.u.) of an ester concentrate daily for a month, with no

Biochem. 1949, 45

other food. Much of the vitamin was excreted, but the liverlevel rose threefold. Some vitamin appeared in the extractsfrom lungs, fat bodies and testes, but not in the skin or theovaries. The experiment indicates poor assimilation, but thismay have been due to the fact that the animals (like allfrogs in captivity) refused to eat spontaneously. Theabsence of vitamin A from the ovaries is consistent with theobservation that it is not detectable in newly spawned eggs.

Fa8ting frogs

The ability to do without food for long periods is a wellknown characteristic of frogs. In an experiment in whichtwenty-five female frogs were kept without food from 18July to 9 December 1947, the fat bodies had been exhaustedand body weights had declined by some 35%. Ovariandevelopment was greatly retarded. The decreases in weightof the viscera during inanition were in general agreementwith previous observations (Ott, 1924). Most of the in-testines contained faeces of liquid consistency with a

'xanthophyll'/carotene ratio of 4/1 as compared with thenormal faecal value of 2. The vitamin A level ofthe liver hadnot fallen very much, but the eyes and the kidneys had beendepleted; 97% of the total vitamin was found in theliver.The results recorded in Tables 16 and 17 need little

discussion. Perhaps the major point is that despite thegreat changes incidental to prolonged fasting the overallX/C quotient for the whole frog rises to 4-6 exactly as innormal animals (3.6-4A6) over the same period of the year.This seems to eliminate dietary influence and supports theidea of selective utilization of different carotenoids.

40

OrganSkinSkinSkinSkinOvariesOvariesOvaLiverMuscleMuscleMuscleOviductTestesFat bodiesSmall intestine

Mono-ester1519151333272018889

22233413

Free272017131513227380848167705581

Group ...

TestesLiverSkinMuscle

B C

C/F(mg./g.)0-325.32-70*77

Fat(mg.)

1-72-29.96-8

Carotenoid(.tg.)0-689.3

286-2

C/F(mg./g.)044-22-909

Vol. 45 625

R. A. MORTON AND D. G. ROSEN

Table 16. Fat, carotenoids and vitamin A infemale frogs keptin captivityfor 25 weeks

(Average decrease in body weight 35%. Means not measured.)

OrganliverEyeKidneySkinOvariesImmature ovariesOviductsImmature oviductsTonguePancreasLungsLeg muscleOesophagusStomachIntestineLarge intestine

Average wt. oforgan as

percentageof body wt.*

0-991-340-239.015-261.198-300-940-830-0250-2749.470-310-750-430-19

* Compare Table 9 column 2.

Percentage of total'xanthophyll' existing as

f ~ ~ ~~A

Di-ester Mono-ester Free form8 18 744 11 8569 19 1225 15 6037 47 1626 60 14

GENERAL COMMENTS

The present study perhaps makes timely a generalcomment on the place of distribution studies in thegrowth of ideas on the biological significance ofparticular compounds. It is a very uncertain place;the pattern of distribution is a resultant of super-imposed patters, of intake, assimilation, storageand utilization, of precursors and metabolites, andof enzyme capacities and 'detoxication' processes.Sometimes distribution studies seem merely to addnew difficulties, but at other times they suggestfruitful lines of attack. A few examples may begiven:

Vitamin A. The distribution data are consistentwith storage in the liver and with localized functionin the eyes, but the apparent absence of vitamin Afrom normal nerve tissue, bone and mucosa is sur-

prising in the light of the pathology of avitaminosisA. Comparative biochemistry adds to tho com-

plexity, e.g. the basking shark is found to store novitamin A in its liver and the tunny fish to storeenormous amounts.

Cholesterol. The concentration of cholesterol in theadrenals pointed to functions, the nature of whichis clarified by the effects of stress or injectionsof adrenocorticotrophic hormone. On the otherhand, the broad picture of cholesterol distributionmerely emphasizes the inadequacy of existingknowledge.Provitamin D. The opcurrence of 7-dehydro-

cholesterol in skin fat is now seen to be significant,but its presence in ripe frog ovaries, in intestinalmucosa and in the sterols from worms is no easier tounderstand than the high concentration of ergo-sterol in yeast.Aswas implied at the outset, thisinvestigationwas

prompted by the idea that carotenoids may havefunctional significance in at least some animals. Theadvantage of the frog is that carotenoids are presentat all stages in the life cycle; its disadvantage is thatpossible clues may be masked by the casual eco-logical variables and by shifting enzyme capaci-ties. In the present state of carotenoid biochemistrythe need is to find a bodily site or a developmentalphase marked out by the distribution as possessingspecial interest.

Research in the field is halted by the lack ofserviceable hypotheses. The present work suggeststhat: (a) beneath much that may be casual there arestrong suggestions of significance; (b) in both malesand females, the sexual cycle exhibits patterns ofstorage, transfer and disappearance of carotene andxanthophyll, consistent with independent andsignificant roles; (c) the whole method of approachis in essence exploratory and its aim is to pro-voke the experiments which turn surmises intocertainties.

626 I949

Vitamin A(.g./frog)

24-70-620-074

Fat(mg./frog)

1-290-721-213.39

17-02-175-19

Carotenoid(Ag./frog)

7.40-5

19-222-18-71-30-30-150-150-134-80-120-40-20-05

3-3

XanthophyllCarotene

1-97

2-092-71

10-219-00-783.374-92

11-0

7-25

1-786-37

Table 17. Distribution of 'xanthophyll' among thefreeform, mono- and di-esters in the organs offa8ted'PlaWyna"I-a

OrganLiverLeg muscleSkinOviductsOvariesImmature ovaries

jemate JrOg8y

Vol. 45 CAROTENOIDS AND VITAMIN A IN THE FROG 627

SUMMARY

1. The occurrence ofcarotenoids and vitaminA infrogs (Rana temporaria) and tadpoles has beenstudied and new metrical data have been obtainedon size and weight.

2. Analytical methods for the accurate and speedydetermination of carotenoids and vitamin A havebeen described and tested.

3. Whole tadpoleswere studied at different stagesof development and the changes in total 'xantho-phyll' and carotene content recorded. The presenceof chlorophyll is ascribed to green food undergoingdigestion. The 'xanthophyll '/carotene quotientdeclines from about 9 to about 3 as developmentproceeds. Vitamin A was detectable in tadpoleeyes.

4. The weights of frog organs expressed as per-centage of body weight are recorded and maxiimumand minimum values given when variations are con-siderable. Much of the carotenoid actually ingestedis contained in undigested food present in the ali-mentary tracts ofinsects or other prey. The frog doesnot ingest preformed vitamin A but converts caro-tene to the vitamin. About 85% ofthe total vitaminoccurs in the liver (mainly as ester); the remainder isnormally about equally divided between kidneys andeyes. Frog kidneys contain a higher proportion ofvitaminA than do mammalian kidneys. Most of the

vitamin A in the eye is ester, stored in the choroid.Carotenoids are dispersed over most organs but quiteunevenly. The deposition of carotenoids in theovaries is so large that a marked difference resultsbetween the sexes in carotenoid storage and utiliza-tion.

5. Mature ovaries contain in relatively high con-centration a substance showing the four absorptionmaxima characteristic of 7-dehydrocholesterol andergosterol.

6. Data have been obtained on the seasonal varia-tion in different organs ofsome or all ofthe following:carotene, 'xanthophyll' (free, mono-ester, di-ester),vitamin A (free and esterified) and total fat. Theresults on normal and fasting frogs reveal: (a) theretention of liver reserves in hibernation and in-anition; (b) well-defined seasonal variations in thecarotenoid storage of different organs; (c) some indi-cation of the continuance of carotenoid metabolismduring hibernation; (d) the overwhelming quanti-tative importance of the ovarian requirements;(e) a drainonvitamin Areserves duringreproduction;(f ) the distinct patterns of storage and utilization ofvitamin A, carotenes, 'xanthophylls' and fat in thefrog.We are indebted to the Medical Research Council and the

Ministry ofFood for financial assistance. One ofus (D.G.R.)held an Evans, Lescher and Webb Studentship and latera Johnston Fellowship in the University of Liverpool.

REFERENCES

Ackermann, J. (1938). Bull. int. Acad. Cracovie, B. ia, 241.Bartz, J. P. & Schmitt, F. C. (1936). Amer. J. Phy8iol. 117,

280.Boyd, E. M. (1938). J. Phy8iol. 91, 394.Brunner, 0. & Stein, R. (1935). Biochem. Z. 282, 47.Comar, C. L. & Zscheile, F. P. (1942). Plant Phy8iol. 17,

198.Dietel, F. G. (1933). Klin. W8chr. 12, 601.Eekelen, M. van (1934). Acta Brev. neerl. Phy8iol. 4, 65.GiUam, A. E. (1938). Biochem. J. 32, 1496.Glover, J., Goodwin, T. W. & Morton, R. A. (1947).

Biochem. J. 41, 97.Harris, D. G. & Zscheile, F. P. (1943). Bot. Gaz. 104, 515.Lederer, E. & Rathmann, F. H. (1938). Biochem. J. 32,

1252.

Lonnberg, E. (1929). Ark. Zool. 21, B (3).Manunta, C. (1934). Atti Soc. Nat. Mat. Modena, 65,

71.March, F. (1937). Proc. zool. Soc. Lond. 107, 415.Morton, R. A. & Stubbs, A. L. (1946). Analyst, 71,

348.Ott, M. D. (1924). Amer. J. Anat. 33, 17.Pepkowitz, L. P. (1944). J. biol. Chem. 155, 219.Rand, C. (1935). Biochem. Z. 281, 200.Smith, C. L. (1947). Private communication.Wald, G. (1935). J. gen. Physiol. 19, 781.Zechmeister, L. & Tuzson, P. (1936). Hoppe-Seyl. Z. 238,

197.Zscheile, F. P., White, J. W. Jr., Beadle, B. W. & Roach,

J. R. (1942). Plant Physiol. 17, 331.

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