556 A. W. PHILLIPS AND P. A. GIBBS 1961 logical properties of peptides derived from casein The authors wish to thank Dr D. A. H. Hearfield for reflected more specific differences in chemical details of the assay and inoculum media, Dr A. J. Woiwod constitution. and Mr R. Knight for constructing the high-voltage power pack, and Miss E. M. Anderson for her able technical STMMARY assistance. 1. Columns of Sephadex G-25 have been used REFERENCES for the fractionation preliminary of tryptic digests Cole, S. W. & Onslow, H. (1916). Lancet, ii, 9. of casein. Gladstone, G. P. & Fildes, P. (1940). Brit. J. exp. Path. 21, 2. Fractions from Sephadex columns have been 161. examined for their ability to stimulate the growth Green, A. A. (1933). J. Amer. chem. Soc. 55, 2331. of a strain of Streptococcus equi8imili8. Maximum Hearfield, D. A. H. & Phillips, A. W. (1961). Nature, Lond., stimulatory activity appeared to be localized in a 190, 266. fairly narrow region of the total peptide material. Ingram, V. M. (1958). Biochim. biophys. Acta, 28, 546. 3. Further fractionation of peptides derived Katz, A. M., Dreyer, W. J. & Anfinsen, C. B. (1959). from casein has been achieved by subjecting frac- J. biol. Chem. 234, 2897. Merrifield, R. B. & Woolley, D. W. (1958). J. Amer. chem. tions from columns of Sephadex to two-dimensional Soc. 80, 6635. separations involving the successive use of paper Porath, J. (1960). Biochim. biophy8. Acta, 39, 193. chromatography and high-voltage electrophoresis. Woiwod, A. J. (1949). J. gen. Microbiol. 3, 312. Over one hundred spots due to peptides have been Woiwod, A. J. (1960). J. Chromat. 3, 278. detected. Yemm, E. W. & Cocking, E. C. (1955). Analy8t, 80, 209.

Bsochem. J. (1961) 81, 556 The Colorimetric Reaction between Vitamin A2 Aldehyde and Antimony Trichloride

BY P. A. PLACK Unit for Biochemical Re8earch bearing on Fi8herie8' Problerrw,* National Inetitute for Re8earch in Dairying, Shinfield, Reading (Received 18 May 1961)

The occurrence of vitamin A1 aldehyde in the EXPERIMENTAL eggs of some marine teleosts has recently been re- ported (Plack, Kon & Thompson, 1959). To extend Preparation of vitamin A1 aldehyde. Vitamin A1 aldehyde this study to the eggs of freshwater teleosts, a was prepared from the crystalline alcohol (Roche Products method for the determination of vitamin A2 alde- Ltd.) as described by Plack et al. (1959). For the pre- hyde was required, since preliminary experiments paration of vitamin A2 aldehyde, the A, aldehyde was purified by chromatography but not crystallized. had shown that the A2 form was present together Preparation of vitamin A2 aldehyde. Vitamin A1 aldehyde with vitamin A1 aldehyde in salmon eggs (Plack, (2.38 g.) was converted into vitamin A2 aldehyde as 1958). Cama et al. (1952) and Farrar, Hamlet, described by Henbest, Jones & Owen (1955). The crude Henbest & Jones (1952) have prepared crystalline preparation in light petroleum (b.p. 40-60°), was adsorbed vitamin A2 aldehyde and, for the coloriinetric re- on a 20 cm. x 4 cm. column of 300 g. of aluminium oxide action with antimony trichloride, gave Am.. 730- (British Drug Houses Ltd., for chromatographic adsorption 740 mg fading to 705 mp, and 730 m,u fading to analysis) weakened by the addition of 30 ml. of water, 705 m,u, respectively. Wald (1938-39) had pre- and the column was developed with 2-5 1. of 2% (v/v) of viously found this to be 705 m,, and Wilt freshly-redistilled diethyl ether in light petroleum (b.p. Am,, 40-60'). A yellow diffuse band was eluted first, followed (1959), with extracts from natural materials, found by a strong orange band, but neither contained vitamin A2 A., to be between 700 and 705 m,u. The study of aldehyde. The main orange band was eluted more slowly the colorimetric reaction between vitamin A2 alde- and was collected in three fractions. From their absorption hyde and antimony trichloride reported here shows curves, fraction 1 contained much vitamin A1 aldehyde and how these differences of Am. may have arisen and, was rejected, but fractions 2 and 3 contained reasonably though not exhaustive, provides the basis for a pure vitamin A2 aldehyde and were combined; the solvent quantitative test. was removed under reduced pressure, and the residue dis- solved in 30 ml. of light petroleum (b.p. 40-60'). Spon- * Grant-aided by the Development Fund. taneous crystallization occurred overnight at - 20°, appar- Vol. 81 DETERMINATION OF VITAMIN A2 ALDEHYDE 557 ently from one nucleus, and the yield was 0-695 g. Two dride, A.R., when required, was added to the solutions at recrystallizations from light petroleum were carried out, and the rate of 2 % (v/v). the last traces of solvent were removed under high vacuum For the reaction with vitamin A2 aldehyde, 2 ml. of the (0-01 mm. Hg), to give a final yield of 0-488 g. The crystals of reagent was rapidly pipetted into a glass cell of 1 cm. light- aldehyde were sealed in 16 evacuated small glass ampoules. path containing 0-5 ml. of a solution of the aldehyde in The crystals were monoclinic, with m.p. 66-68' (Kofler CHC13, and the cell was quickly transferred to the spectro- apparatus), unchanged by further recrystallization. Both photometer for measurement. Cama et al. (1952) and Farrar et al. (1952) reported melting Antimony pentachloride. The diphasic system of Briugge- points of 77-78°, although one preparation (Cama et al.) mann, Kraus & Tiews (1952) was used, and the reagent was had m.p. 61°. With the exception of m. in chloroform, prepared as described by them from solid SbCl,, K2Cr2O7 values for the absorption characteristics of my preparation and conc. HCI. One drop (about 0-05 ml.) was added to (Table 1) were in good agreement with those given by 3 ml. of the aldehyde solution in dry, ethanol-free CHCl3 in Farrar et al. (1952) and in fair agreement with the values a stoppered flask, the flask was shaken gently for 1 min. obtained by Cama et al. (1952). and the contents poured into a spectrophotometer cell for A sample of crystals of m.p. 77-79° was recently received measurement. Most of the aqueous phase remained in (a gift from F. Hoffmann-La Roche and Co. Ltd., Basle, the flask. Switzerland); with the Kofler apparatus its m.p. was found Solutions of commercial SbCl5 (British Drug Houses Ltd.) to be 75-77°. A solution in light petroleum of the crystals 15, 1-5 and 0.15% (v/v) in CHCl3 were also used, 1 drop of m.p. 66-68° was seeded with the higher-melting-point together with 1 drop of acetic anhydride being added to material and gave crystals of m.p. 75-77°. A solution of 2 ml. of the aldehyde solution in CHCl3. these last crystals was cooled to - 200 and seeded with Glycerol dichlorohydrin. 1:3-Dichloropropan-2-ol, for material of m.p. 66-68°, when two types of crystal forma- vitamin A determination, was obtained from Kodak Ltd. tion were produced: a few clusters with a purple tinge, and used as supplied, since preliminary tests only were m.p. 75-770, and a larger quantity of orange-red clusters, carried out. To 2 ml. of the reagent in a spectrophotometer m.p. 66-68°. cell, 0-5 ml. of the aldehyde solution in CHCl3 was added, The vitamin A2 aldehyde crystals of m.p. 66-680 were the two solutions were mixed with a Polythene rod and the used in the experiments reported here, but the quantitative cell was transferred to the instrument. aspects of the reaction were checked with crystals of m.p. Spectrophotometers. Most measurements of light-absorp- 75-77 tion were carried out with an Optica CF4 double-beam A solution of the aldehyde in hexane was isomerized with recording spectrophotometer, equipped with a red-sensitive 12 (see Fisher, Kon & Plack, 1957). The extinction was about DuMont photomultiplier. This instrument was used in one 5 % lower, and A., about 1 m,u lower, after isomerization, of three ways: (a) to give a plot of percentage transmission suggesting that the original aldehyde was all-trans rather against time after addition of reagent for one particular than a cis-isomer. Solutions in CHC13 of the aldehyde were wavelength; (b) to give percentage transmission against stable for many weeks when kept in brown-glass containers wavelength; (c), a combination of (a) and (b), with the in a dark cupboard at room temperature. chart running continuously to give a time scale, and the Antimony trichloride. Two preparations were used. The region of maximum absorption scanned in rapid succession, solution of SbCl3 in CHC13 supplied by British Drug Houses so that changes of 4. with time could be followed. Ltd. for vitamin A tests, containing about 14% (w/w) of Readings in terms of percentage transmissions were con- SbCl3, will be referred to as SbCl3 solution (commercial). verted into extinction values, and in some later experi- Solutions prepared by distilling solid A.R. SbCl3 (British ments extinction values were plotted directly. Drug Houses Ltd.) and collecting the middle fractions in a After Ama.. had been determined with the recording flask containing a weighed quantity of CHC13 (used as spectrophotometer, more exact readings of extinction were supplied by May and Baker Ltd.) will be referred to as obtained with fresh samples in a Beckman model DU purified SbCl3 solution. Initially, the CHCl3 was purified quartz spectrophotometer, or in the manual version of the by washing with water, to remove ethanol and decompose Optica CF4 spectrophotometer. any COC12, and drying over anhydrous Na2SO4. Solutions of SbCl3 in this purified CHC13 frequently gave an oily deposit, which disappeared on heating but returned on RESULTS cooling. Such a deposit was not produced when CHC13, as Reaction with antimony trichloride supplied, was used. These solutions prepared in the labora- tory were usually diluted to contain 14% (w/w) of SbCl3, Commercial solutions and solutions of purified but other concentrations were also tested. Acetic anhy- antimony trichloride. When antimony trichloride

Table 1. Absorption characteristics of the preparation of vitamin A2 aldehyde (m.p. 66-68°) compared with those of preparations obtained by other workers Plack, this paper Cama et al. (1952) Farrar et al. (1952) __ A Solvent Ama. (m) El/m Ama. (mv) El%/m Amax, (mj) El% * Chloroform 410 1280 407 1360 407 1295 Ethanol 400 1370 397 1410 400 1350 Hexane 387 1440 385t 1490t 388: 14604 * Calculated from values for c. t In light petroleum (b.p. 40-60°). t In pentane. 558 P. A. PLACK 1961 solution (commercial) with acetic anhydride was used as the reagent, solutions of vitamin A., alde- oo cq Eoob hyde gave a green-blue colour, the Am.. of which varied with the concentration of aldehyde. In one -o instance, a reaction mixture containing 2-05 ,tg. of aldehyde/ml. had A 733 m,u, and one containing 10 t- 'IO CO 0-205 had A,, 705 m,. Similar variations 04cs_em ,tg./ml. o eq cO CO were found with all batches of antimony trichloride solution (commercial), but different batches gave different values of A... for the same solution of aldehyde. They also required different times to 00 "000 reach E..., the variation being from 30 sec. to Di 2 min. 30 sec., and gave different rates of fall of CO OD d L with time. The colour reactions ofthese various

O O 4D <:> 00 x O batches of antimony trichloride solution (com-

q " mercial) with vitamin A1 alcohol were perfectly normal. Antimony trichloride was therefore purified by d, o acm_o4 t oo _0~ XCOrce distillation, and dissolved in chloroform. A 14 % 0z oc o I solution in the presence of acetic anhydride gave no improvement in the variations of A,,, (Table 2). Ca "RC s > oost-0-I-. rO c to0 Effect of acetic anhydride. With a 14 % solution ,o 3 omme of purified antimony trichloride, the effect of < C OD.. - qP4 0l4 acetic anhydride was studied. In Table 2 are shown the values for A,. for different concentra- tions of vitamin A. aldehyde at different times after i~~~~( to m- o1 P- "q r- P- o I s the reagent and aldehyde solutions had been mixed. In one experiment, acetic anhydride was added to Cs the reagent, in the other it was omitted. Only when acetic anhydride was absent were the values for .$ _ ~~ ~~Ob'oect m (M M(M c 0 CB el lo A,. constant (741 m,u; range, in this and other cotPa ce cs _ c - q O*4 0 0o experiments, 739-743 m,u) irrespective oftime after mixing and concentration of vitamin A. aldehyde. This result was reproducible with all the batches of .B .0 14% purified antimony trichloride solution pre- oq pared. The same experiments were done with vitamin A1 aldehyde, and, both in the presence and absence of acetic anhydride, Am.. was constant with time ~ . 0 c_ eq CwO 1* I* and with concentration ofaldehyde. The two values _~~~~~~oD D o c - were slightly different, however, being 661 m,u 0s 6) ~eq (range 659-663 mp) with added acetic anhydride 0;l eq eq eq r eq and 666 mu (range 665-667 m,u) in its absence. A commercial solution of antimony trichloride, (D_ R. uCv C m qIto with no added acetic anhydride, still gave varia- a) P _. C CO- tions in A,,, of from 738 to 730 m, with final con- centrations of vitamin A. aldehyde of from 1-98 to 0-198 ,ug./ml. Purification of the reagent and omission of acetic anhydride were therefore both necessary for stability of A.,. G4 _ aC- 10-1 Acetic anhydride had no effect on the ultra- e q>v e violet curve of vitamin A1 aldehyde or A aldehyde. Effect of concentration of antimony trichloride. >3oo ao ' M N N Table 3 shows the effect of different concentrations CO toq 10 XC e Cq CO of purified antimony trichloride with a single con-

o eq eq eq: centration of vitamin A, aldehyde in the absence of acetic anhydride. Values for E1l. were nearly constant over the range 10-16 % of antimony tri- Vol. 81 DETERMINATION OF VITAMIN A2 ALDEHYDE 569 Table 3. Effect of concentration of purifled antimony chloride, but at concentrations above and below trichloride on the colorinetric reaction with vitamin As they were lower. Since the absorption maximum aldehyde was broad, values for E1°'4 at Am. would not be The reaction mixture contained 0-5 ml. of a solution of significantly different from those given at 741 m,. vitamin A, aldehyde in CHC13 (7.16 ,ug./ml.) and 2 ml. of a A,m, tended to increase with concentration of anti- solution of purified SbCl, in CHC13, to give a final concen- mony trichloride, but was reasonably constant over tration of 1*43 jug. of aldehyde/ml. E.,,= (741 m1L) was the range 10-20 %. The period of stability of E... measured with the manual Optica spectrophotometer and varied with concentration of trichloride, but the El% calculated from the mean of three measurements; the maximum stability, at concentrations of 20 and time factor and AmaX. were determined with the Optica 25 % (approx. 40 sec., Table 3), was associated with recording spectrophotometer. values of E'% below the maximum. The stability Concn. of E,.. varied slightly with different preparations, of SbCl, Time after and it is probably also dependent on the rate of solution mixig when mixing the reagent and the test solution, and on added El%. Em". steady Amax (%, w/w) (741 mju) (sec.) (mIA) temperature. At 200, a saturated chloroform 5 3500 20-25 738 solution of anti- 10 4330 20-35 742 mony trichloride contains about 20 % (w/w), so that 12 4400 20-35 742 the 25 and 30 % solutions were supersaturated. At 14 4400 30-45 742 lower the commercial 16 4300 40-75 743 temperatures, solutions of 20 4030 50-90 743 about 14 % sometimes crystallize out. It seemed 25 3770 20-60 745 sensible from the result of this experiment to use 30 3840 20-45 744 the same concentration of antimony trichloride used for other purposes, namely 14 % (w/w). 07 Quantitative a&pect8 of the reaction. With a 14 % solution of purified antimony trichloride and no acetic anhydride, the maximum value of the ex- tinction at 741 m,u increased linearly with concen- tration of vitamin A, aldehyde up to 10 pg./ml. (final concentration of aldehyde in reaction mix- ture, 2 ,ug./ml.). A mean value for E1l% (741 mjp) of 4200 (range 4000-4400) was obtained with five preparations of antimony trichloride solution and solutions of vitamin A, aldehyde prepared from four separate weighings. With these same solutions, A,,. varied over the range 739-743 my. Solutions of the crystals of m.p. 75-77' gave values within these ranges. Concentrated solutions of the aldehyde, m.p. 66-68', gave an initial fleeting red colour in this reaction, which immediately changed to green-blue. Fig. 1 shows a series of curves drawn by the re- cording spectrophotometer at various times after mixing chloroform solutions of vitamin A, aldehyde and purified antimony trichloride. They are not true absorption curves, since during the time of scanning one curve (35 sec.) the extinction diminish- ed slightly. With the high speed of scanning used, 560 600 640 680 720 760 800 840 there is also a slight displacement of A,, to longer Wavelength (m,u) wavelengths. As the extinction at A.. diminished, the shoulder at about 700 m, became more pro- Fig. 1. Curves drawn by the recording spectrophotometer nounced. for the green-blue colour produced by the reaction of 0.5 ml. of a solution of vitamin A, aldehyde in chloroform Reaction with antimony pentachloride (7-16 jig./ml.) and 2 ml. of a 14% solution of purified antimony trichloride in chloroform, showing the fading of Preliminary experiments with antimony penta- the colour with time. The peak of curve (a) was traced chloride in a diphasic system showed that the Am., 45 sec. after the reaction had begun, (b) 2 min. 7 sec., of the green-blue colour varied from 705 to 724 mA (c) 6 min. 22 sCC., (d) 10 min. 35 sec. and (e) 14 min. 38 sec. with increasing concentrationIs of vitamin A, alde- Speed of scanning 8 mj4Lsec. hyde. The rate of fading was rapid and the specific 560 P. A. PLACK 1961 extinction coefficient not so great as with antimony sample of crystalline vitamin A2 aldehyde was pre- trichloride. Antimony pentachloride in chloroform pared. Henbest et al. (1955) were unable to obtain gave no colour reaction, though with the 15 and crystalline material by their method, but I was 1-5 % (v/v) solutions the yellow colour of the fortunate enough to obtain spontaneous crystal- aldehyde disappeared. lization which gave a product of m.p. 66-68°, con- siderably different from the two values previously Reaction with glycerot dichlorohydrin reported, 77-78° (Farrar et al. 1952; Cama et al. Vitamin A2 aldehyde gave a blue-green colour 1952) and 610 (Cama et al. 1952), and yet from its with glycerol dichlorohydrin, rising to a maximum absorption characteristics apparently pure all- intensity about 2 min. after mixing, and starting to tran8-vitamin A2 aldehyde. Vitamin A1 aldehyde is fade a few seconds later. With final concentrations known to exist in dimorphic forms, with m.p. 57 of aldehyde in the reaction mixture of 0-27- and 650 (Robeson, Blum, Dieterle, Cawley & 4-33 ,zg./ml., A.. of the coloured product was con- Baxter, 1955) and it may be that all-tran8-vitamin stant at 618 m,. Over this range of concentrations, A2 aldehyde exists in three different crystalline the colour intensity was proportional to the con- forms. centration of aldehyde, and the mean Elm The reactions of solutions of the crystalline alde- (618 mI,) was 1340. Vitamin A1 aldehyde with hyde with antimony trichloride in the presence of glycerol dichlorohydrin gave a coloured product acetic anhydride gave, even with purified materials, with Ama. 653 mp, with a shoulder at 610 m,. green-blue colours whose A, varied with concen- tration of aldehyde. This variation (Table 2) pos- DISCUSSION sibly accounts for the discrepancies in A.. reported in the literature. Two other colour reactions were Vitamin A and its related compounds are usually tested in-an effort to overcome this variation. With determined either by measuring their absorption antimony pentachloride, in a diphasic system, there of light in the ultraviolet or by a colorimetric was still variation of A, with concentration of reaction, of which the Carr-Price test (Carr & aldehyde; antimony pentachloride in chloroform Price, 1926) with antimony trichloride is the most produced no colour with vitamin A2 aldehyde, widely used. Each method has certain advantages, although Murata & Nagashima (1960) have recently but in this Laboratory we prefer the colorimetric used this reagent successfully for determination of technique, since it is more sensitive and less subject vitamin A1 alcohol. With glycerol dichlorohydrin to interference when small quantities of vitamin A ks did not vary with concentration of aldehyde, in extracts from natural materials are to be but vitamin A1 aldehyde gave a higher Am^X than measured. For the determination of vitamin A2 vitamin A2 aldehyde and, for the latter, the specific aldehyde, there is another reason for choosing a extinction coefficient was low compared with that colorimetric method. The aim was to devise a in the Carr-Price test; the reagent therefore seemed method for the determination of vitamin Al and A2 less suitable than antimony trichloride, provided aldehydes in the presence of each other (see Plack that the irregularities with the latter could be & Kon, 1961); in the ultraviolet, the wavelengths overcome. of their respective absorption maxima vary accord- Further experiments with purified antimony tri- ing to the solvent used, but are separated by only chloride showed that omission of acetic anhydride about 20 m,u, whereas in the colour reaction with from the reaction mixture led to values of Ama that antimony trichloride the difference between their were constant over a range of concentrations of maxima is much greater, 75 m,u in the absence of vitamin A2 aldehyde and remained stable for at acetic anhydride, so that the detection of the two least 5 min. (Table 2). Acetic anhydride is usually aldehydes in mixtures is easier and the deter- added in the antimony trichloride reaction to re- mination of each more accurate with this test. move any traces of water and avoid clouding, but The Carr-Price test with antimony trichloride Carr & Price (1926) in their original communication is applied in slightly different ways in different do not mention its use, and in the reaction reported laboratories; the amount of acetic anhydride used here it is essential to omit it. The omission did not varies, and so does the proportion of reagent to lead to any cloudiness in the solutions. sample; the instrument available for measuring the Conditions have thus been found for a quanti- colour intensity may use a small or broad band tative colorimetric test for vitamin A2 aldehyde width and may determine whether the maximum with antimony trichloride. The wavelength of intensity can be read or whether a reading at some maximum absorption is not affected by changes in fixed time after mixing reagent and sample must aldehyde concentration, the colour intensity is be used. Extinction coefficients must therefore be stable for a short time, and varies linearly with the determined for the conditions used, and for the concentration of vitamin As aldehyde. With the purpose of defining them for this Laboratory a range of values for El°om (4000-4400, mean 4200) Vol. 81 DETERMINATION OF VITAMIN A2 ALDEHYDE 561 found for different preparations of antimony tri- 4. Some observations on antimony penta- chloride, the characteristics of a particular pre- chloride and glycerol dichlorohydrin (1:3-dichloro- paration should be determined with pure vitamin propan-2-ol) as colorimnetric reagents for vitamin A2 aldehyde for accurate work, but for determin- As aldehyde are presented. ationsofthealdehydeinnatural materials, the mean value of 4200 can be used. The variations in A... (739-743 mru, mean 741 m,u) are not significant REFERENCES since, with the broad maximum of the absorption Bruggemann, J., Kraus, N. & Tiews, J. (1952). Z. anal. curve, values of extinction over this range would Chem. 135, 241. be essentially the same, within the experimental Cama, H. R., Dalvi, P. D., Morton, R. A., Salah, M. K., error of the test. Steinberg, G. R. & Stubbs, A. L. (1952). Biochem. J. 52, 535. SUMMARY Carr, F. H. & Price, E. A. (1926). Biochem. J. 20, 497. 1. Crystalline vitamin A2 aldehyde with m.p. 66- Farrar, K. R., Hamlet, J. C., Henbest, H. B. & Jones, 680 has been from vitamin A1 E. R. H. (1952). J. chem. Soc. p. 2657. prepared aldehyde. Fisher, L. R., Kon, S. K. & Plack, P. A. (1957). Proc. Roy. 2. With purified antimony trichloride, 14 % (w/w) Soc. B, 147, 200. in chloroform containing 2 % (v/v) acetic anhydride, Henbest, H. B., Jones, E. R. H. & Owen, T. C. (1955). the wavelength of maximum absorption of the J. chem. Soc. p. 2765. green-blue colour produced with vitamin A2 alde- Murata, T. & Nagashima, J. (1960). J. Vitaminol. 6, hyde increased with concentration of the aldehyde 158. and decreased with time. Plack, P. A. (1958). Rep. nat. In8t. Dairy., Reading, 3. In the absence of acetic anhydride, the wave- p. 112. length of maximum absorption of the colour pro- Plack, P. A. & Kon, S. K. (1961). Biochem. J. 81, 561. Plack, P. A., Kon, S. K. & Thompson, S. Y. (1959). duct was steady at 741 m,i, the colour developed Biochem. J. 71, 467. remained at full intensity for about 15 sec., the Robeson, C. D., Blum, W. P., Dieterle, J. M., Cawley, J. D. maximum extinction varied linearly with concen- & Baxter, J. G. (1955). J. Amer. chem. Soc. 77, 4120. tration of aldehyde and E1:,,, (741 mi/) was 4200 Wald, G. (1938-39). J. gen. Phy8iol. 22, 391. (range 4000-4400). Wilt, F. H. (1959). Developmental Biol. 1, 199.

Biochem. J. (1961) 81, 561 A Comparative Survey of the Distribution of Vitamin A Aldehyde in Eggs

BY P. A. PLACK AiD S. K. KON Unit for Biochemical Research bearing on Fi8herie8' Problem8,* National Institute for Research in Dairying, Shinfield, Reading (Received 18 May 1961) We have recently reported the occurrence of was present (Plack, 1958), but a study ofthe eggs of vitamin Al aldehyde in the ripe eggs of several freshwater and anadromous fish was held up for marine teleosts (Plack, Kon & Thompson, 1959) lack of a suitable method of determining vitamin and in hen's eggs (Plack, 1960). Pollard & Bieri A1 and A2 aldehydes in the presence of each other. (1959) confirmed generally our early findings, but The two aldehydes can be determined by a simple found vitamin A2 aldehyde in the eggs of American extension of the colorimetric reactions of the two 'herring' from Chesapeake Bay, and Winterstein & compounds with antimony trichloride, but this Hegedus (1960) confirmed our finding with hen's method could only be applied accurately when the eggs. With cod, the aldehyde is found only in eggs anomalous behaviour of vitamin A2 aldehyde in the from ripening ovaries (Plack, Woodhead & Wood- colorimetric reaction had been clarified by Plack head, 1961) and from our experience it is so with (1961). other fish. With the method now available, a comparative Some preliminary experiments with ripe eggs survey of the occurrence of vitamin Al and A4 from salmon suggested that vitamin A2 aldehyde aldehydes in eggs was made. The aldehydes were found in all the ripe vertebrate eggs so far ex- * Grant-aided by the Development Fund. amined, but not in eggs from five invertebrates. 36 Bioch. 1961, 81