Biochem. J. (1969) 112, 231Printed in Great Britain

Reactions of Vitamin A with Acceptors of ElectronsINTERACTIONS WITH IODINE AND THE FORMATION OF IODIDE

By J. A. LUCY* AND F. ULRIKE LICHTItStrangeway8 Re8earch Laboratory, Cambridge

(Received 11 October 1968)

1. The reactions of retinol and retinoic acid with iodine were investigated sinceknowledge of the chemical reactions of vitamin A with acceptors of electrons mayshed light on its biochemical mode of action. 2. Colloidal retinol, but not retinoicacid, reacts with iodine to yield a blue-green complex that rapidly decomposes,giving iodide and an unknown species with Amax. at 870mit. 3. In addition, bothretinol and retinoic acid reduce iodine to iodide by a reaction that does not involvean intermediate coloured complex; this reaction appears to yield unstable car-bonium ion derivatives of the vitamin. 4. The presence of water greatly facilitatesthe production of iodide from vitaminA and iodine. 5. Possible chemical pathwaysinvolved in these reactions are discussed. 6. It is suggested that the chemicalproperties of retinol and retinoic acid that underlie their biochemical behaviourmight be apparent only when the molecules are at a lipid-water interface, and thatvitamin A might be expected to react with a number of different electron acceptorsin vivo.

It is reported in the preceding paper (Lichti &Lucy, 1969) that electron transfer can occur whenretinol, or retinoic acid, interacts with 7,7,8,8.tetracyanoquinodimethane or with chloranil (tetra-chloro-1,4-benzoquinone), and that the radicalanions of 7,7,8,8-tetracyanoquinodimethane andchloranil are formed under appropriate conditions.The present paper reports some reactions ofvitaminA with iodine, which include reduction of iodine toiodide. Molecular iodine is known to form charge-transfer complexes with many different donors ofelectrons (Andrews & Keefer, 1964) and, on

complete electron transfer, the I- ion is produced.In our investigations particular attention has beenpaid to the reactions of vitamin A with iodine thatoccur when water comprises the bulk environment,since the amphipathic nature of retinol that isresponsible for its surface-active (Bangham, Dingle& Lucy, 1964) and membrane-active (Lucy, 1964)properties indicates that the vitamin may belocated at a lipid-water interface in vivo.The present findings on the chemical behaviour

of vitamin A are thought to be consistent with thepossibility that the vitamin may be involved inelectron-transfer reactions in vivo (Dingle & Lucy,1965). If vitamin A is involved in electron-transfer

* Present address: Department of Biochemistry, RoyalFree Hospital School of Medicine, University of London,London W.C.l.

t Present address: Molecular Biology Laboratory,University of Wisconsin, Madison, Wis. 53706, U.S.A.

reactions in vivo, the widespread biological actionsof the vitamin (Moore, 1957) may arise from itsinteractions with one or more acceptor moleculesin biochemical processes occurring on, or in,different membranes.A preliminary communication has been published

(Lucy & Lichti, 1967).

MATERIALS AND METHODS

Reagent8. Synthetic crystalline retinol (Roche ProductsLtd., Welwyn Garden City, Herts.), retinoic acid (Distilla-tion Products Industries, Rochester, N.Y., U.S.A.) and I2(AnalaR; British Drug Houses Ltd., Poole, Dorset) were

used without further purification. All aqueous solutions ofI2 were freshly prepared. The concentrations of I2 in theaqueous solutions used were determined by completelyextracting the 12 into light petroleum followed by spectro-photometric measurements of the I2 in the organic solvent.Retinol was handled as described by Dingle & Lucy (1962).

Spectrophotometric mea8urement8. Most of the spectrawere obtained by means of a Unicam SP.700 recordingspectrophotometer. All spectral measurements were madeat room temperature.

Uptake of OH- ion8. Consumption of OH- ions was

measured by means of a Radiometer automatic titrator,used as a pH-stat at pH7-0, with standardized 0-01 M-NaOHas the titrant. In procedure A, retinol and iodine in ethanolwere added separately under N2 to previously boiledphosphate buffer (0.5 mm). Since a small uptake of baseoccurred with I2 alone, the I2 was added first and the pHbrought to 7 0 before addition of retinol. The rate ofaddition of base levelled off about 3-4min. after addition of

231

J. A. LUCY AND F. U. LICHTIthe retinol; consumption of OH- ions/mole of retinol wascalculated for the plateau region. In procedure B, solutionsof retinol and I2 in ethanol were mixed, and a sample of themixture was added to the phosphate buffer. To correct forthe base uptake due to I2 alone, a separate titration wasmade of I2 added to the buffer from ethanol. All titrationswere made at room temperature.

RESULTSInteraction8 ofretinol and iodine in water. Addition

of a fresh solution (6 ml.) of iodine in water (80,ug./ml.) to ethanol (0.02 ml.) containing retinol (20mg./ml.) immediately yields a dark precipitate: theheterogeneous reaction mixture appears blue byreflected light and dark green by transmitted light.A comparable result is obtained if the iodinesolution is added to a fresh colloidal suspension ofretinol in water, prepared by rapidly adding water(5ml.) to ethanol (0.05ml.) containing retinol(20mg./ml.). It appears that, whichever of thesetwo techniques is employed, the iodine reacts withcolloidal aggregates of dispersed retinol to form thecoloured complex, since the complex is not formedwhen retinol and iodine are mixed in ethanol.Neither is the coloured substance formed whenretinol and iodine are allowed to interact in ethanoland then subsequently added to water (see below).Some degree of micellar organization of retinolmolecules may therefore be a prerequisite for theformation of the coloured complex.No coloured precipitate is produced with retinol

when sufficient potassium iodide is added to theaqueous iodine solution to destroy the absorptionband at 460m,u of iodine in water. In the presenceof excess of iodide, the iodine-containing solutionexhibits only the intense absorption bands at 286m,uand 354m,u due to the I3- ion (cf. Robin, 1964). Itwould thus seem that free iodine is necessary forthe formation of the coloured precipitate withretinol.The coloured heterogeneous reaction mixture

produced on the interaction of iodine with colloidalretinol initially has a pronounced absorptionmaximum at about 610mu (16.4kcyc./cm.) (Fig. 1).This absorption band is presumably due to thepresence of a new molecular species, since theabsorption maxima of iodine and of retinol inwater are at 460m,u and 325m,u respectively andneither substance has an absorption band in the600-900m,u region (Fig. 1).

Investigation of the ultraviolet absorption of thereaction mixture in the 320-330m,u region, wherecolloidal retinol has its maximum, reveals that theabsorption due to retinol is considerably decreasedwithin 1 min. of addition of iodine; the position ofmaximum absorption also shifts to about 295m,u(34keyc./cm.). The spectrum of the reactionmixture then remains relatively unchanged during

1-0 -

08 F

E 0 61

0 4K'

0 2

0 ....................

20 5 10 kcyc./cm.500 667 1000 m[L

Fig. 1. Spectral changes with time (in minutes) ofa mixtureof I2 (0-3mM) and retinol (0.24mM) in water ( ). Theaqueous I2-retinol reaction mixture was prepared byadding 50Sp. of a solution of I2 (60mM) in ethanol to 10ml.of N2-saturated water; this was then added to 50/41. of asolution of retinol (48mM) in ethanol. Correspondingspectra of retinol (0-24mM) (----) and of I2 (0-3mM)( ) in N2-saturated water are also shown. The spectrumof the I2 solution is stable for at least 30min.

the succeeding 30min. (Fig. 2). A decrease inextinction and changes in the shape of the spectrumof colloidal retinol occur during the autoxidationof retinol by molecular oxygen in an aqueousenvironment (Fig. 2). The changes occurring withmolecular oxygen involve the development of finestructure in the spectrum of the dispersed retinol(Lucy, 1965), however, and they differ from thoseobserved in the reaction of retinol with iodine inwater (Fig. 2). Nevertheless a shift ofthe absorptionmaximum to about 295 m,u also occurs in theautoxidation reaction after a relatively long time(1-2 hr.) (J. A. Lucy, unpublished work).

Decomposition of the retinol-odine complex. Thevisible colour of the retinol-iodine complex fadesquite quickly, and spectral studies reveal that thedecomposition of the complex is accompanied bythe development of new absorption bands inthe ultraviolet and near-infrared regions of thespectrum.

Iodine in water has an absorptionmaximum in theultraviolet at about 205 m,u (48.8 kcyc./cm.); its

232 .1969

1-2 r

REACTIONS OF VITAMIN A WITH IODINE

I*o

0 8

0 6

E

0 4

0 2

0

50 40 30 20 kcyc./cm.200 250 333 500 m

Fig. 2. Spectrum of the reaction mixture ( ) containingI2 (0-044mM) and retinol (0.026mm) 30min. after mixingin air-equilibrated water. Spectra of retinol (0.026mM)1 min. and 30min. after suspending in air-equilibrated water(----) and of I2 (0.044mm) in air-equilibrated water(. ) are also shown. The spectrum of the I2 solution is

stable for at least 30min.

extinction at 226m,u (44.3keyc./cm.) is 25% ofthat at 205m,u. Within 1min. of preparing thereaction mixture of iodine and colloidal retinol, theiodine peak at 205 m,u is no longer apparent. Duringthe first minute a shoulder develops at about 226 m,t(44.3kcyc./cm.) and a distinct new maximum ispresent at this wavelength after 30min. (Fig. 2). Asecond new maximum is also observable at about195min (51keyc./cm.). Control experiments showthat none of these changes occurs either with a

solution of iodine in water or with colloidal retinol(which has a minimum at 226m,u) over a 30min.period (Fig. 2). Since the I- ion in water is knownto have maxima at 194m,L and 226m,u (Franck &Scheibe, 1928), these observations indicate thatiodine is reduced to iodide as the retinol-iodinecomplex decomposes.Experiments were carried out to compare the

rate of decay of the extinction of the colouredcomplex at 610m,u and the formation of the peakattributed to iodide at 226m,u. The reactionmixture was placed in a cm. cuvette, and thedecrease at 610m,u was followed continuously for15min. The increase at 226m,u was determinedsimilarly in an experiment in which a 1mm.

cuvette was employed. The rates of change at both

wavelengths were most rapid during the initial5min. (Fig. 3). Although the shapes of the twocurves of Fig. 3 would seem to be consistent withthe interpretation that iodide is derived from thecoloured complex of retinol with iodine, experi-ments described below indicate that iodide canalso be formed by a pathway that is independent ofthe coloured complex.

If iodide is formed from the retinol-iodinecomplex, equivalent cationic material is presumablyproduced simultaneously. It is therefore note-worthy that a new species, which absorbs at870m,u (11*5keyc./cm.), is formed in addition toiodide as the coloured complex decays (Fig. 1). Ina control experiment the absorption due to retinoldispersed in water was observed to increasegenerally in the 800-90Om,u region, apparently as aresult of a change in the state of aggregation of thedispersed retinol. No maximum developed at870m,u, however, in the absence of iodine. Anisosbestic point is formed at about 740m,u(13.5kcyc./cm.) as the retinol-iodine complexdecomposes (Fig. 1) and this may indicate that thematerial absorbing at 870m, is being produceddirectly from that absorbing at 610mu, butno firm conclusions can be drawn in view of theheterogeneous nature of the reaction mixture.Nevertheless, the formation of iodide and theproduction of the species absorbing at 870m,u bothoccur most rapidly during the 5min. periodimmediately after the interaction of iodine withcolloidal retinol (Fig. 1). It is suggested thereforethat the band at 870mg may be due to the positivecounterion that is formed from the retinol-iodinecomplex at the same time as the iodide.

Recovery of iodine. Since the above spectralstudies indicated that the iodine of the complex israpidly reduced to iodide, experiments were madeto recover iodine after reoxidation of the iodide.Samples of the reaction mixture containing theretinol-iodine complex were extracted once withlight petroleum at different times after formation ofthe complex, and the recovered iodine was estimatedby means of its characteristic absorption at 525m,in light petroleum. Repetition of the extractionprocess yielded no further iodine from individualsamples and, after one extraction, the aqueoussamples gave no colour on acidification and treat-ment with starch. A blue colour (Amax. 615mp)formed in the presence of acid and starch, however,when the iodide present in these solutions wasoxidized by sodium nitrite. A comparison of theprogressively decreasing quantities of free iodinerecovered with the increasing densities of colourobserved in the starch test with sodium nitrite isshown in Fig. 4 for periods up to 10min. after theinitial formation of the retinol-iodine complex.The blue starch-iodine reaction can be used,

Vol. 112 233

J. A. LUCY AND F. U. LICHTI6

1-4

1-2

a 1-00.C

0-8

10

9

0 2 4 6 8 10 12 14Time (min.)

Fig. 3. Changes in extinction with time (in minutes) of the reaction mixture ofretinol (0-025mM) and I2 (0-66mM)in air-equilibrated water. The extinction at 610mu ( ) was measured in a 1 cm. cuvette, and that at 225 m,u(----) in a 1 mm. cuvette.

0 2 4 6 8 l0Time (min.)

Fig. 4. Comparison of the rate of consumption of I2(extinction at 525 mit; x ) with the rate of formation ofI- ions (extinction at 615m,i; *) as indicated by thestarch-iodide reaction, when retinol reacts with iodine.The aqueous I2-retinol reaction mixture was prepared byadding 15 ml. of a freshly prepared solution of I2 (0.48mM)in air-equilibrated water to 60j1. of retinol (70mM) inethanol.

within appropriate concentration limits, as a

measure of the iodide produced from the inter-action of retinol with iodine. In two separatedeterminations, 1.0 and 1-5moles of iodine were

consumed/mole of retinol (iodine being present

initially in a slight molar excess). After 10min.2-0 and 1-3 moles of iodide/mole were found to havebeen produced. A detailed quantitative analysisof this system was not made, however, in view ofthe likelihood (considered below) that iodide isproduced by two simultaneous but independentreaction pathways that yield respectively either1 or 2 moles of iodide/mole of iodine consumed.Formation of iodide in the ab8ence of the coloured

retinol-iodine complex. Water greatly facilitatesthe production of iodide from iodine and retinol.Thus the I- ion is apparently not formed, or atleast is formed much more slowly, when retinol andiodine are mixed in ethanol rather than in water, asindicated by the slow increase in the ultravioletabsorption spectrum at 222-227m,u (44-45kcyc./cm.) (Fig. 5). Nevertheless, the absorption of thereaction mixture in ethanol (0-02mM-retinol and0-04mM-iodine) undergoes a marked decrease withtime in the region of the spectrum where retinolabsorbs maximally (325mu or 30-8kcyc./cm.).After 40min. the absorption maximum has shiftedto about 295m,u (34kcyc./cm.) and this mayindicate that an addition reaction of iodine with adouble bond of retinol has occurred. These spectralchanges occur much more rapidly if the reactionis carried out at higher reactant concentrations(lOmM).When retinol and iodine are allowed to interact

for a short time in ethanol at concentrations of10mM, and the reaction mixture is then diluted1000-fold with water, the spectrum due to iodide

234 1969

REACTIONS OF VITAMIN A WITH IODINE

E

45 40 35 30 25 20 kcyc./cm.222 250 286 333 400 500 mjA

E

45 40 35 30 25 20 kcyc./cm.222 250 286 333 400 500 ml,

(c)

45 40 35 30 25 20 kcyc./cm.222 250 286 333 400 500 rnF

Fig. 5. Spectral changes with time (in minutes) during the reaction of retinol (0 021 mm) with I2 (0-040mm) in(a) ethanol, (b) aq. 50% ethanol and (c) water. , Reaction mixture; , solution of I2 (0O040mM) 1 mi.after preparation; ......solution or dispersion of retinol (0*021 mM) 1 min. after preparation. The spectra of the I2and retinol controls changed only slightly during 40min., since all experiments were done in N2-saturated solvents.

appears immediately but no coloured complex isobserved. This experiment indicates that theproduction of a coloured complex is not essentialfor the process of electron transfer from retinol toiodine.

It is noteworthy that, after lmin. in aq. 50%(v/v) ethanol, absorption maxima are apparent at295, 323 and 339m,u (34.0, 31-0 and 29.5kcyc./cm.)and a shoulder is observable at 308m,u (32.5 kcyc./cm.) in addition to the iodide peak at 226m,u(44-3kcyc./cm.). After 40min. the iodide peak hasslightly increased. The multiple absorption bandsin the 300-345mp, region have virtually dis-appeared by this time, leaving a prominentabsorption maximum at about 295m,u (34kcyc./cm.). Since the latter band and that due to iodide,but not the multiple bands between 300m,u and345 m,u, are formed within 1 min. when retinolreacts with iodine in water (Fig. 5), it seemspossible that the multiple bands noted in aq. 50%ethanol may be due to an intermediate that isproduced in the electron-transfer process and thatis more stable in ethanol-water than in water alone.

Retinoic acid and iodine. Retinoic acid dispersedin water has a broad absorption maximum at about370mt, (27kcyc./cm.) and, apart from a decrease inextinction and a further increase in the width ofthe band, the spectrum of the dispersed materialremains unchanged for at least 20min. at roomtemperature. Dispersed retinoic acid does not give

a coloured complex with iodine comparable withthe retinol-iodine complex. Failure to form such acomplex may be due to the difficulty of preparingsufficiently concentrated dispersions of retinoicacid, the acid being more difficult to disperse thanretinol.

Iodine nevertheless reacts readily with retinoicacid in water as is shown by the marked decreasewithin 1 min. of the absorption due to retinoic acidat about 370m,u and the appearance of a band atabout 226m,u corresponding to the I ion (Fig. 6).As with retinol and iodine, new material with aAmax. at about 295m,u is also formed. The develop-ment of the band at 295m,u can be accelerated bypre-mixing retinoic acid and iodine (at concentra-tions of 10mM) in ethanol and then diluting themixture 1000-fold with water. When retinoic acidand iodine interact in aq. 50% ethanol, the 1 ionis again rapidly produced, although its appearanceis slightly slower than in water alone. With aq. 50%ethanol new peaks also develop between 294 and345m,u (34 and 29kcyc./cm.) (Fig. 6); the behaviourof retinoic acid and iodine therefore resembles thatof retinol and iodine at this concentration of water.Further, as with retinol, iodide does not seem to beproduced when retinoic acid and iodine react inethanol, although interactions nevertheless occurthat lead to the formation from retinoic acid ofmaterial absorbing at shorter wavelengths (295 m,).

Oon8urnption of OH- ion8. As in the reactions of

Vol. 112 235

J. A. LUCY AND F. U. LICHTII.1

.0

0-9

0 8

0 7

E

0 6

0'5

0 4

0 3

0-2

0.1

45 40 35 30 25 20 kcyc./cm.222 250 286 333 400 500 mp

0 71

E

0 6

0 5

0 4

0 3

0 2

0o

400 500 m1"

(c)

45 40 35 30 25 20 kcyc./cm.222 250 286 333 400 500 mp1

Fig. 6. Spectral changes with time (in minutes) during the reaction of retinoic acid (0.020mm) with I2 (0.040m2)in (a) ethanol, (b) aq. 50% ethanol and (c) water. -, Reaction mixture; ----, solution of '2 (0.040mM) 1mi.after preparation; ...... solution or dispersion of retinoic acid (0.020mm) min. after preparation. The spectra

of the I2 and retinoic acid controls changes only slightly during 40min., since all experiments were done inN2-saturated solvents.

vitamin A with 7,7,8,8-tetracyanoquinodimethaneand chloranil reported in the preceding paper(Lichti & Lucy, 1969), consumption of OH- ionsis a feature of the interactions of both retinol andretinoic acid with iodine. Uptake of OH- ions wasmeasured in two different ways. In method A,vitamin A in ethanol was added to iodine dissolvedin dilute buffer of the desired pH; in method B,vitaminA and iodine were mixed in ethanol and thereaction mixture was then added to the buffer.Method A allows colloidal aggregates of retinol toform when the retinol is added to the bufferediodine solution, and in this procedure the colouredretinol-iodine complex is formed. In method B an

interaction between retinol and iodine occurs inthe ethanol, as indicated by the observed spectralchanges that are reported above. Apparently as a

result of this interaction the coloured retinol-iodinecomplex is not produced when the ethanolic reactionmixture is subsequently added to water. Which-ever procedure is used, the pH of the buffer falls toa low value (approx. pH 3) by the time the sampleaddition is complete, and about 1 min. elapsesbefore the pH is fully restored to its initial value bythe titrator. The rapid fall in pH indicates thateither an acid is being formed or some other reactionis occurring that liberates protons, such as thereaction of a carbonium ion with water. From a

Table 1. Uptake of OH- ions a80ociated with thereaction of retinol and iodine at pH7-0

Uptake of OH- ions was measured with a Radiometerautomatic titrator, used as a pH-stat at pH 7-0 as describedin the Materials and Methods section. In series A, vitamin Aand I2 were added separately to the dilute buffer; in series Bvitamin A and I2 were premixed in ethanol and the mixturewas added to the buffer. The values in parentheses in thefifth column represent the numbers of determinations.

Concn. Conen. I2/retinolof I2 of retinol molar

Procedure (mm) (mm) ratioA 0-52 0-52 1.0A 1-04 0-52 2-0A 1-56 0-52 3-0A 1-72 0-151 11-8A 1-815 0-120 15-1A 1-815 0-060 30-3B 0-52 0-52 1-0B 1-04 0-52 2-0B 1-77 0-15 11-8

OH- ionsconsumed

(moles/mole ofretinol)

1-3+0-1 (3)2-0+0-1 (3)2-6+0-3 (3)4-8+0-2 (3)6-8 (1)6-7 (1)1-6+0-1 (3)3-4+041 (3)6-7+0-6 (3)

consideration of the known reaction of electrondonors (see the Discussion section) it is thought thata carbonium ion may be involved.

In a series of experiments in which the ratio of

E

236 1969

REACTIONS OF VITAMIN A WITH IODINE

iodine to retinol was varied, the quantity of OH-ions consumed/mole of retinol was observed todepend on the molar ratio ofthe reactants (Table 1).The finding that at least six OH- ions can beconsumed/molecule of retinol appears to imply thatthe electrons of at least three double bonds ofretinol are available for transfer to iodine when theratio of acceptor to donor molecules is very high.The decreased consumption of OH- ions in

method A as compared with method B (Table 1) isconsidered to result from the formation of thecoloured complex between retinol and iodine inmethod A. It is suggested that the colloidalaggregates of retinol produced in method A mayreact with iodine by two competing pathways. Inone reaction retinol forms an unstable colouredcomplex, whereas in the other it may form acarbonium ion derivative that reacts with water toconsume OH- ions. Formation of the colouredcomplex would thus decrease the quantity ofretinol available for carbonium ion formation. Inprocedure B apparently only the carbonium ionpathway is followed. With retinoic acid con-sumption of OH- ions was more comparable inmethods A and B. Thus in experiments withiodine (0-59mM) and retinoic acid (0.15mM), themean values of 3 0 + 0-2 and 3-4 + 0-1 moles of OH-ion consumed/mole of retinoic acid were obtainedfrom three determinations each by methods A andB respectively. This is consistent with the fact thatretinoic acid does not form a coloured complex withiodine.

After the initial readjustment of the pH, uptakeof OH- ions continued very slowly for 10-20min. inmethod A but not in method B. This behaviourwas more pronounced with retinoic acid than withretinol, and it is therefore unlikely to be associatedwith the decay of the coloured retinol-iodinecomplex. The slow consumption of OH- ions mayoccur because method A allows the formation ofaggregates of retinol molecules that, if they are notinvolved in the coloured retinol-iodine complex,may react relatively slowly with iodine to yieldcarbonium ions. This interpretation would appearto be supported by the noticeably slower con-sumption of OH- ions in experiments with retinoicacid and iodine, since retinoic acid gives much largeraggregates than retinol in water.

DISCUSSION

A number of substances resemble retinol informing blue complexes with iodine (Cramer, 1955).In their early studies on the interactions ofsaponarinand cholalic acid with iodine Barger & Field (1912)concluded that no substance that is dissolved asseparate molecules will give this reaction; the'typically colloidal reaction' of benzacridines and

acridines with iodine to give blue or brown colours(Kermack, Slater & Spragg, 1930) has also beenreferred to by Albert (1966) as evidence thatsolutions of the ions ofmany acridines may containdimers or highly aggregated micelles. The forma-tion of the coloured complex of iodine with retinolmay thus depend partly on the ability of retinolto form colloidal aggregates in aqueous dispersions(cf. Straus, 1939; Lucy, 1965). By contrast retinoicacid, which does not form a blue complex withiodine, cannot be dispersed in water at concentra-tions equal to those used with retinol since grossaggregation occurs. Cramer (1955) suggested thatthe most important factor contributing to theformation of blue inclusion compounds containingiodine, such as the starch-iodine complex, may beelectronic interactions of the donor-acceptor typebetween the host molecule and iodine, and Szent-Gyorgi (1960) has discussed the formation of a bluecomplex of iodine with cortisone (Amax. 740m,u) interms of charge transfer. Thus a second factorcontributing to the formation of a coloured complexof retinol with iodine is probably the electron-donorcharacter of retinol.The instability of the blue retinol-iodine complex

apparently stems from the strength of the donorproperties of retinol, since our observations indicatethat iodine is reduced to iodide as the colouredcomplex decays. If the coloured material isequivalent to a charge-transfer complex, of thetype [retinol . I2], dissociation may subsequentlyyield an ionic complex of the type [retinol.I]+I-,in which the cation [retinol. I]+ absorbs at 870m,t.This dissociation would resemble that ofthe complexbetween pyridine and iodine, which gives rise to the'inner complex' of N-iodopyridinium iodide,containing the cation (C6H5 . NI)+ (Reid & Mulliken,1954). It is relevant that infrared-absorptionbands due to (y-picoline2. I)+ and I3- ions havebeen observed with polar solvents in addition tothe band associated with the charge-transfercomplex between y-picoline and iodine (Haque &Wood, 1967). The production of iodide from thecharge-transfer complex formed between triphenyl-arsine and iodine has also been interpreted interms of the formation of an inner complex con-taining I- ion (Bhat & Rao, 1966).The interactions between retinol and iodine that

we have observed have some resemblance to thosebetween ,B-carotene and iodine reported by Lupinski& Huggins (1962) and Lupinski (1963). In polarorganic solvents ,B-carotene reacted with iodine togive material having a new absorption band at10OOm,u; new bands also appeared at 290 and360m,, which were attributed to the 13-ion. It hasbeen proposed by Lupinski (1963) that the solid fi-carotene-iodine complex, C40H56,2I2, is best repre-sented by the structure (C4OH56......I+)I3-, in

Vol. 112 237

J. A. LUCY AND F. U. LICHTI

C6H4 *N(CH3)2- I\ - +/0H3

CH3/ - CH

C6H4 *N(CH3)2(I)

(CH3)2N\ /N(CHa)2 NC\ /CNCHsNC\NC +2 C- C

(CH3)2N/ N(CH3)2 NC/ -\CN(III) (IV)

N I OH2*OH

(CH3)2N\+ +/N(CH3)2 NC\. _ CN- > C-C + 2 C-C\

(CH3)2N/ N(CH3)2 NC/ CN

(II)

CH3C/CH2.OH

/--+\ C H C>II +

(V)

CH3

C NI C. H2OH

+ + 2H20-+

(V)

CHS

1 CH2.OHs~~~~UOHCH +2H+OH

(VI)

which the cation contains an iodonium ion ratherthan a carbonium ion. Ebrey (1967) has suggestedthat the band at 10OOm, observed by Lupinski(1963) is due to ,-carotene that has had its absorp-tion band shifted by the interaction with iodine(cf. Platt, 1959), and he has proposed that the com-plex may have the structure I3..-.. C+ :[C-C=]n * -

C .C... I+. Since both the band at 10OOm,uobserved by Lupinski (1963) with ,-carotene-iodine, and that at 870m,u found in the retinol-iodine system in the present experiments, are about540m,u from the normal absorption bands of,B-carotene and retinol respectively, a similarinterpretation may apply to the decomposition ofthe initial coloured retinol-iodine complex. On thisbasis retinol may have mediated a dissociation oftheneutral iodine molecule into the iodonium ion, I+,and the iodide ion, I-, by virtue of forming a

relatively stable 'trimolecular charge-transfer com-plex' with the two ions (Platt, 1959). Since vitaminA would not be consumed in such a process, a

reaction of this type, which may be regarded as a

catalytic reaction, might be of significance inrelation to the mechanism of the systemic actionsof the vitamin in vivo.Our observations on the production of iodide in

the absence of a coloured intermediate complexindicate the existence of a second mechanism forthe production of iodide from vitamin A andiodine. This would appear to involve a direct

electron transfer from vitamin A to iodine, and maybe similar in principle to the reaction of tetrakis-(p-dimethylaminophenyl)ethylene with iodine(Anderson, Elofson, Gutowsky, Levine & Sandin,1961) in which two I- ions are formed and a dication(I) is produced that contains two positively chargednitrogen atoms. Since a double bond in retinol (orretinoic acid) is presumably the source of theelectrons that are transferred from the vitamin toiodine, the electron-deficient product will probablycontain one or two carbonium ions. The reactionproduct may be comparable in this respect with thedicarbonium ion (II) formed in the reaction oftetra(dimethylamino)ethylene (III) with tetra-cyanoethylene (IV) (Wiberg & Buchler, 1963).

Ifthe dication (V) were produced on the reductionof iodine by retinol, it would be expected to reactwith water or added OH- ions (cf. Sorensen, 1965)to yield the polar product 13,14-dihydroxyretinol(VI), which has one double bond less than theoriginal molecule. Since compound (VI) has fourconjugated double bonds, it would be expected tohave an ultraviolet-absorption maximum at about290m,u. It is therefore noteworthy that at leasttwo OH- ions are consumed/molecule of retinol,and that material absorbing at about 295m, isproduced when retinol and iodine, or retinoic acidand iodine, react in the presence of water. It seems,however, that more than one double bond of retinolis apparently available for electron transfer since

+ 2 I-

238 1969

REACTIONS OF VITAMIN A WITH IODINE

the observed consumption of OH- ions indicatesthat more than two carbonium ions are formed/molecule of retinol in the presence of excess ofiodine.Although a mechanism for the formation ofiodide

involving direct electron transfer from vitamin A toiodine would be consistent with the behaviour ofvitamin A towards TCNQ that is reported in thepreceding paper (Lichti & Lucy, 1969), it must notbe overlooked that loss of one double bond fromretinol by any means will yield a product having anabsorption maximum at about 295m,. Thus theproduct of an addition reaction of iodine with adouble bond would be expected to absorb in thisregion of the spectrum. Iodine does not reactreadily with unsaturated olefinic systems, however,since unlike the addition of chlorine and brominethe addition of iodine is normally reversible and theposition of the equilibrium lies well to the side offree olefins (De La Mare & Bolton, 1966). It remainspossible, nevertheless, that iodine may interactwith retinol by an electrophilic addition reaction toyield a carbonium ion intermediate >C(I)-C+< thatmay react rapidly with the aqueous solvent to givethe grouping>C(I)-C(OH)<; this behaviourwould becomparable with that observed when the morereactive bromine molecule interacts with olefinicacids in water (Read & Read, 1928). In thisinstance, the number of moles of OH- ionsconsumed/mole of retinol will equal the number ofmoles of iodine reacting. By contrast, the numberof moles of OH- ions consumed/mole of retinol willbe twice the number of moles of iodine reacting inan electron-transfer reaction such as that yieldingthe compounds (V) and (VI). Although thereaction of some molecules of vitamin A withiodine by an electrophilic addition reaction cannotbe excluded in our studies, particularly with retinoicacid, the consumption of 1-6 and 3-4 moles of OH-ions/mole of retinol in experiments in which themolar ratios of iodine/retinol were 1-0 and 2-0respectively (Table 1, procedure B) would not seemto be explicable solely in terms of electrophilicaddition. Furthermore, the electrophilic additionof iodine, followed by a reaction of carbonium ionintermediates with the solvent, cannot account forvalues that are greater than 5 0 for the number ofmoles of OH- ions consumed/mole of retinol.The development of spectral fine structure in the

305-340m,u region that we observed when retinoland iodine interacted in aqueous 50% ethanol, andthe subsequent appearance ofa single band at about295m,u, parallel the spectral changes noted instudies on the autoxidation of colloidal retinol insodium chloride solution (Lucy, 1965). Since therapid autoxidation of retinol and the developmentof fine structure within 1 min. does not occur in thepresence of aqueous 50% ethanol (Lucy, 1966)

similar products may be formed when retinol reactswith iodine in aq. 50% ethanol and when colloidalretinol is autoxidized by molecular oxygen insodium chloride solution, especially as a rapid fallin pH occurs both in the autoxidation reaction(J. A. Lucy & F. U. Lichti, unpublished work) andwhen retinol reacts with iodine.

Derivatives of retinol having the retro configura-tion exhibit spectral fine structure at a relativelylong wavelength as compared with the parentcompound (e.g. retinyl acetate has one absorptionband at 325 m,,; retro-retinyl acetate has threebands at 333, 348 and 367m,u; Beutel, Hinkley &Pollak, 1955). The development of multiple bandsat about 310, 325 and 340m,u in experiments withretinol may therefore indicate that a rearrangementofdouble bonds has occurred, and the position ofthemultiple bands could indicate that one double bondhas been destroyed. As discussed in the precedingpaper (Lichti & Lucy, 1969), loss of a single electronfrom retinol may give a radical ion grouping of thetype >C+-C <. By analogy with the work of Blatz &Pippert (1968) on the carbonium ion formed byprotonation of retinyl acetate, the loss of a singleelectron from retinol might thus yield resonanceforms such as (VIIa) and (VIIb); the reaction ofstructure (VIIb) with water would then give ahydroxylated radical (IX) containing a doublebond adjacent to the ring as in the retro series ofcompounds. It is relevant that Beutel et al. (1955)have postulated that the conversion of vitamin Ainto retro-vitamin A under acid conditions mayproceed via the intermediate 'conjugated acid'(VIII). If structure (VIIb) were a more stableresonance form than structure (VIIa), the majorintermediate reaction product would be the radical(IX) and this might exhibit spectral fine structure.Since it is known that organic radicals can behaveas electron donors (Buley & Norman, 1964), afurther electron-transfer reaction in which theradical (IX) is the donor may then occur and giverise to the structures (Xa) and (Xb). If structure(Xa) were more stable than structure (Xb), thefinal product, after reaction with water, would be13,14-dihydroxyretinol (compound VI, see above),which would absorb at a shorter wavelength thanretinol and, like retinol, it would have no finestructure.

Isenberg & Baird (1962) have remarked that theenhancement of charge transfer and free-radicalformation by polar solvents may have biochemicalimplications. They also suggested that, since theformation of free radicals in biochemical systemstakes place in an aqueous environment, inter-actions with the medium may be expected to playa significant role. Such considerations are par-ticularly apposite in relation to vitamin A, whichhas surface-active and membrane-active properties.

Vol. 112 239

J. A. LUCY AND F. U. LICHTI

CH3

0~CH2.OHCH1VIIa +

(VIIa)

IS *CH2OH

(VIII)

OH

(IX)

CH3

p/,<~NC,,<\CHCH.OH

OH

(Xa)

f - 1t/~~~CH-C2.OHHOH

(Xb)

In the present experiments the characteristicspectrun of the I- ion did not develop at all rapidlywhen retinol interacted with iodine in ethanolcontaining less than 5% of water. The observationsreported in the preceding paper (Lichti & Lucy,1969), that an aqueous sodium chloride medium isapparently necessary for successful electron transferfrom retinol to chloranil, provide another exampleof the effect of environment on retinol. This is alsoseen in the autoxidation of retinol by molecularoxygen, which occurs most readily in aqueoussodium chloride, less readily in water and muchmore slowly in organic solvents (Lucy, 1965, 1966).The influence of the environment in these reactionsleads us to suggest that the chemical properties ofretinol and retinoic acid that are responsible for theirbiochemical behaviour might become apparentonly when the molecules are at an interface betweenlipid and water. In such a situation molecules ofvitamin A make contact with the aqueous phasebut simultaneously retain the properties that theyexhibit in a non-polar environment. It is note-worthy that retinal in aqueous 2% digitoninbehaves as if it were in a somewhat non-polarenvironment with regard to the generation offree radicals by incident light (Grady & Borg,1968). It may also be relevant to the biochemistryof retinol that the rates of bimolecular chemical

reactions occurring in micellar systems can beincreased by including both reactants in or on themicelle (cf. Bruice, Katzhendler & Fedor, 1968).Our findings on the effect of the environment on

retinol contrast with those published by Bhowmik,Jendrasiak & Rosenberg (1967) on charge-transfercomplexes of lecithin with iodine. These workersobserved the formation of I3- ions when iodineinteracted with lecithin in water or in carbontetrachloride, and when lecithin was in the solidstate. Wobschall & Norton (1967) have concluded,however, that water is necessary for the formation ofthe blue complexes of certain steroids with iodine.The experiments reported in the present paper

indicate that the donor reactions of vitamin A invitro are not restricted to reduction ofsubstances like7,7,8,8-tetracyanoquinodimethane and chloranil,which, when they are reduced to their respectiveradical anions, accept a total of only a singleelectron. In addition both observations (F. U.Lichti & J. A. Lucy, unpublished work) on theautoxidation of retinol in an aqueous environment(Lucy, 1965, 1966) and the studies of Grady & Borg(1968) on light-induced free radicals of vitamin Aindicate that one molecule of the vitamin may serve

as a donor ofan electron to a second molecule undercertain circumstances. It is suggested that thislack of specificity may possibly be of biological

240 1969

/CH2-OH+

(VlIb)

Vol. 112 REACTIONS OF VITAMIN A WITH IODINE 241

significance, since it might enable the vitamin toreact with a number of different acceptors indifferent environmental situations in vivo.

During this work J. A. L. was a member of the externalstaff of the Medical Research Council; F. U. L. was a post-doctoral fellow of the U.S. National Institutes of Health.We thank Mrs D. Shelford for technical assistance.

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