June, 19611 2,2-DICHLOROPROPIONATE (DALAPON SODIUM SALT) Reactions of Steroids Sulphuric Acid (the with Acetic Anhydride 373 and Liebermann - Burchard Test) BY R. P. COOK (Biochemist~y Department, Queen’s College, University of St. Andrews, Dundee) The intensities of colours developed and the rates of reaction when various steroids (CIS to C,,) dissolved in acetic acid were treated with acetic anhydride and sulphuric acid (the Liebermann - Burchard test) have been investigated ; cholesterol was used as standard. Only certain sterols (C,,, C,, and C,J and some of their derivatives gave a typical blue-green colour. The colour intensities produced , relative to that developed by cholesterol, could be grouped in five classes: (i) zero or slight, (ii) about half, (iii) equal to and either “fast” or “slow acting,” (iv) about twice and either “fast” or “slow acting” and (v) four to six times greater, all being “fast acting.” The nature and conformation of the substituents in positions 3 and 7 of the sterol ring appear to be important.THE Liebennann - Burchard test consists in adding to a sterol, dissolved in a suitable solvent, acetic anhydride containing a small amount of sulphuric acid, when a (usually) blue-green colour develops; a history of the test was given by Dam.l The test, used both qualitatively and quantitatively, is much employed in studies of sterols, particularly with cholesterol. This paper describes the comparative results obtained after testing a number of steroids and their derivatives dissolved in acetic acid, cholesterol (cholest-5-en-3/3-01) being used as an arbitrary standard.Some observations on cholesteryl esters dissolved in chloroform (many are insoluble in acetic acid) are also made. The colours developed after 1-5 (“fast acting”) and 35 minutes (“slow acting”) were observed and measured (cf. paper by Moore and Baumann2).374 COOK: REACTIONS OF STEROIDS WITH ACETIC ANHYDRIDE [Vol. 86 NAMING OF COMPOUNDS The generic name "steroid" is used for compounds containing the perhydrocyclopenteno- phenanthrene nucleus with various substituents attached. In Table I (p. 375), the main headings are- Sterols-These are steroids having a side-chain of eight, nine or ten carbon atoms, always with a hydroxyl group at position 3. In their systematic description, to avoid use of a large number of roots, cholestane has been taken as the parent compound; to this have been added (as prefixes, with the number in the steroid skeleton or in the side-chain) methyl, ethyl or hydroxyl substituents.Stavtols-These are fully saturated sterols. Stevtols-These are sterols having one double bond and one hydroxyl group in position 3. Di~~droxystenols-These are sterols having one double bond and two hydroxyl groups. Stenadi(tri)e72oZs--These are sterols having two or three double bonds and one hydroxyl group. Compounds defined by a page reference to Fieser and Fieser's "Steroids" are described Systematic naming is generally in accordance with I.U.P.A.C. in more detail in that book.3 rules*; the trivial name or names follow the systematic name. METHOD REAGENTS AND C O M P O U N D S All chemicals used were of analytical-reagent grade, and attention was paid to the removal of all but traces of water.The compounds tested were obtained from the sources listed in the Appendix, p. 380. Cholesterol standard-Commercial cholesterol was recrystallised from glacial acetic acid by Fieser's method and then recrystallised from methanol; the purified compound was stored in dark bottles and kept at -15" C. Its melting-point was 149.5" to 150" C in a sealed capillary tube and 148" to 149' C in a Kofler block; its specific rotation, [ ~ r ] ~ ~ " , was -3943" (C = 1.5 in chloroform). PROCEDURE- As only small amounts of certain compounds were available, samples of about 1 mg were accurately weighed in a tared 1-ml calibrated flask on a microbalance (sensitivity 0.01 mg).Glacial acetic acid was then added, with warming if necessary to ensure solution, and the contents of the flask were diluted to the mark. The cholesterol standard was similarly treated. The colour-forming reagent consisted of chilled acetic anhydride (19 volumes) to which was added 1 volume of concentrated sulphuric acid; the reagent was freshly prepared for each test. For the test itself, 1-0 ml of colour-forming reagent was added to 0.5 ml of steroid solution, and the solution was well mixed with a fine glass rod. The colour developed was observed visually after 1.5 and 35 minutes and after 20 hours. The intensity of colour was measured with an E.E.L. colorimeter (Evans Electroselenium Ltd.), the No. 205 red filter being used.2" C in tubes shielded from direct light. Duplicate determinations were always made, and any discrepant values were re-determined. The results were calculated from the optical densities on a weight-to-weight relationship ; the intensity of colour developed after 35 minutes by 0-5 mg of cholesterol was taken as 100. A numerical relationship was found, but, as the compounds had perforce to be tested in small batches over a period of 3 years, it was thought that a system of intensity rating indicating a broad comparative basis was more suitable than a whole-number relationship. With most of the compounds, the relationship is approximately equimolecular, notable exceptions being bi-steroids (see Table I, samples Nos. 54, 55 and 56) and cholesteryl esters.Colour was developed at 20" RESULTS STEROLS AND RELATED COMPOUNDS- to similar studies on certain of the samples have been included. The results for these substances are shown in Table I. For ease of presentation, referencesTABLE I REACTIONS OF STEROLS AND RELATED COMPOUNDS IN THE LIEBERMANN - BURCHARD TEST Sample No. Stanols- 1 2 3 4 5 6 7 8 9 10 Stenols- 11 12 13 13A 14 15 16 17 The notation used for intensity of colour is based on the optical density compared with that developed by 0-5 mg of cholesterol (taken as 100) : 0 = no reaction; S = 10 to 20; 0-5 C = 40 to 60; C = 80 to 140; 2 C = 160 to 240. Intensities greater than 400 are expressed to the nearest whole-number multiple of C. Colours and intensities after 20 hours are given only when stability or intensification was observed; in general, the colours faded to a green-blue of intensity about 0.5 C Compound 5a-Cholestan-3a-01 (epicholestanol) 5a-Cholestan-3 /3-01 (cholestanol, dihydro- 5 P-Cholestan-3a-01 (epicoprostanol) 5 /3-Cholestan-3 /3-01 (coprostanol, copro- 4a-Methyl-5a-cholestan-3 /3-01 4,4-Dimethyl-5a-cholestan-3 p-01 (di- 5a-Cholestane-3/3, 5a-diol 5a-Cholestane-3 /I, 7a-diol 3-acetate 5a-Cholestane-3 /3, 7 /3-diol 3-acetate 5a-Cholestane-3 /3, 5a, 6/3-triol cholesterol) sterol) meth ylcholestanol) Cholest-4-en-3 /3-01 (allocholesterol) Cholest-5-en-3a-01 (epicholesterol) Cholest-5-en-3 /3-01 (cholesterol) Cholesterol hydrate 19-Norcholesta-6-en-3 /3-01 (norcholesterol) 5a-Cholest-6-en-3 p-01 acetate 5wCholest-7-en-3 /3-01 (lathosterol, 5 /3-Cholest-7-en-3/3-01 (coprostenol) A'-cholestenol) Source No. * 9 9 9 6, 13, 22 14, 28 26 4 1 1 11 15 15 See text 6 29 12 9 2 Colour and relative intensity after- 1.6 minutes 36 minutes r - 0 Brown-green; S None None 0 Pale blue; S 0 Green-blue; 0.5 C 0 Green; S 0 Green-blue ; S 1 Blue-green ; C Blue; C Deep blue-green ; 2 C Blue-green ; 2 C None None 0 Green; 2 C 0 Blue-green ; 2 C 0 Green; 2 C 0 Blue-green ; C 0 Blue-green ; C 0 Pink Blue-green ; C Blue; 6 C Blue-green ; C Blue; 6 C Purple; C Purple; C w 6 E Keferencet + U +I Remarks G n 3a axial 3 #I equatorial J 3a equatorial 313 axial Intensifies to about 0.5 C on standing - 3/3 equatorial, 5a axial - 3 #I equatorial, 5a and 6 j? axial 3 #I equatorial - 3 /3 equatorial - greater than for cholesterol - 3 /3 equatorial - 315 axial - w Z 3/3 equatorial, 7a axial I W c 38 and 78 equatorial } w 0 X - cl - 3a axial Intensity about 10 per cent.W 7 38 equatorial 6 - * See Appendix, p. 380. t See reference list, p. 380.TABLE 1 ( c o d . ) Colour and relative intensity after- w 4 b, SamDle Source NO. Compound Stenols 18 19 20 21 22 23 24 25 (c0Tttd.)- 4a-Methyl-5a-cholest-7-en-3 /3-01 (metho- 4a-Methyl-5a-cholest-8-en-3 /3-01 4a-Methyl-5a-cholest-8( 14)-en-3 /?-01 4a-Methyl-5a-cholest-14-en-3 8-01 4,4-Dimethylcholest-5-en-3/3-01 24a-Methylcholest-5-en-3 8-01 (campesterol) 24b-Ethylcholest-5-en-3 /I-01 (/3-sitosterol) 24-Ethylidenecholest-5-en-3 /3-01 (fucosterol) stenol, lophenol) Dihydyoxystenols- 26 Cholest-4-ene-3/3, 6/3-diol 27 Cholest-5-ene-3 /3, 4/3-diol 28 Cholest-B-ene-3/3, 7a-diol 29 Cholest-5-ene-3 /3, 7 /I-diol 30 Cholest-5-ene-S/?, 24b-did 31 Cholest-B-ene-3j3, 25-diol 32 Cholest-5-ene-3/3, 26-diol Sknadi (tvi) ends- 33 Cholesta-5,7-dien-3 j-01 (7-dehydrochole- sterol) 34 Cholesta-5, 22-dien-3 8-01 (22-dehydro- cholesterol) 35 Cholesta-5,24-dien-3 8-01 (24-dehydro- cholesterol, desmosterol) 36 Cholesta-5,25-dien-3 8-01 37 Cholesta-8(9),22-dien-38-01 (zymosterol) 38 24b-Methylcholesta-8( 14) ,22-dien-3 8-01 39 24b-Methylcholesta-5,22-dien-38-01 40 24b-Methylcholesta-6,7,22-trien-3 /3-01 41 24b-Ethylcholesta-6,22-dien-3~-01 42 24b-Ethylcholesta-7,22-dien-3 p-01 ( A8 ( la) -ergos t erol) (brassicasterol) (ergosterol) (stigmasterol) (a-spinasterol) No.* 15, 28 14 14 14 4 1 1 1 21 12 12 12 9 9.24 18 20 3 3 3 1 4 1 20 1 1 1.5 minutes Deep blue; 5 to 6 C Blue-purple; 2 C Blue-purple ; 2 C, Purple; 3 C Blue; 2 C 0 Green; S 0 Dark blue; 6 C Blue; 2 C Blue-green; 6 C Blue; 6 C 0 0 0 Blue; 6 C 0 0 0 Blue-green; C Blue; 2 C Green; S Blue; 6 C 0 Blue; 5 C Remarks 35 minutes Deep purple; 4 t o 5 C Blue-purple; 2 C Purple; 3 C Green-blue ; 3 C Blue-green ; C Green; C Green; C 3/3 equatorial 3 /3 equatorial 3 /3 equatorial 3 /3 equatorial Green; 7 C Green; 2 C Blue-green; 6 C Blue; 6 C Green; C Green; C Blue-green ; C 3 /3 equatorial, 6 /3 axial 3 /3 equatorial, 4 /3 axial 38 equatorial, 70( axial 313 and 78 equatorial 3/? equatorial 3 /3 equatorial 3 /3 equatorial 3 13 equatorial Green; 4 C Blue-green; C Green; 0.5 C Green; C 3 equatorial 3 /3 equatorial 3 /? equatorial Blue-green; C Green-blue; 2 C Green; C Green; 4 C Blue-green; C 3/3 equatorial Blue-green; 2 C 3 8 equatorial Reference? 8 9 - 6 - 10 - cholestenol 6 (for analogues) - 6 6 0 0 0 w * See Appendix, p.380. t See reference list, p. 380.Sample No. Compound 0 x 0 compounds- 43 44 45 46 47 48 49 50 50A 51 Ketols- 52 53 5a-Cholestan-3-one (cholestanone) 5 8-Cholestan-3-one (coprostanone) Cholest-4-en-3-one (A4-cholestenone) Cholest-5-en-3-one (As-cholestenone) Cholest-7-en-3-one (A7-cholestenone) Cholesta-3,5-dien-7-one Cholesta- 4 6 -dien- 3-one 24b-Methylcholesta-4,7,22-trien-3-one (ergosta-4,7,22-trien-3-one) Enol acetate of sample No. 50 Cholest-4-en-6 8-hydroperoxy-3-one ( A4-cholestene-3-one-6 /3-hydroperoxide) Cholest-5-en-3 8-01-7-one (7-oxo(keto) - Cholest-5-en-3 b, 26-diol- 16,22-dione cholesterol) (kryptogenin) Bi-steroids- 64 Cholestenone pinacol 56 3,3-Bis(cholesta-3,5-dienyl) 66 7-Oxocholesteryl acetate pinacol MisGe~~ane0U.s- 57 Cholest-5-en-3 p-thiol (thiocholesterol) 68 Cholesteryl methyl ether 69 Dicholesteryl ether 60 “Isocholesterol” 61 4,4,14-Trimethy1cho1est-8-en-3 /3-01 ( As-lanosterol, dihydrolanosterol) TABLE I (contd.) Colour and relative intensity after- Source f A \ No.* 1.5 minutes 35 minutes 9 None None 6 Purple: 4 C Blue-green; 4 C 4 0 Pale green; S 4 0 Apple green; 0.6 C 4 Fluorescent green; Intense fluorescent 2 c green; 2 C 4 Fluorescent green ; Intense fluorescent 0.5 C green; 2 C 9 None None 4 None None 4 None Red-yellow fluores- cence; S 4 Purple: 2 C Blue-green; 3 C 4 Purple; C Dark blue; 2 C 4 Green; 0.6 C Ydlow; s 19 0 Blue-green; 2 C 6 0 Blue-green ; C 7 None None 6 Yellow-green Yellow-green fluorescence fluorescence 1 Yellow-green Yellow-green fluorescence fluorescence Remarks Referencet I I - - I - Colour constant after 18 hours - Multiply by 2 for molar comparison - - - Mixture of trimethylsterols - - 3 (p.235) 11 (preparation and structure) - - - - - 3 (p. 364) - * See Appendix, p. 380. t Sep reference list, p. 980.378 [Vol. 86 OTHER STEROIDS- Representative steroids only were tested, but particular attention was paid to available compounds having a A5, 3/3-01 configuration (i.e., 3/3-hydroxyld-en). The sources of these samples are listed in the Appendix, p. 380. Oestrogens-These have eighteen carbon atoms and are described on p. 463 of v steroid^."^ Some samples of oestrone, oestradiol and oestriol from sources Nos.16 and 25 tested under the standard conditions gave an immediate eosin-like fluorescent colour that later faded to yellow, but other samples gave negative results. With the latter, it was observed that a colour could be produced by adding excess of concentrated sulphuric acid. Androgens-These have nineteen carbon atoms and are described on p. 519 of e steroid^."^ 17/3-Hydroxyandrost-4-en-3-one (testosterone) from source No. 17 gave no colour ; 3p- hydroxyandrost-5-en-17-one (dehydroepiandrosterone) from source No. 15 gave a pale yellow ; androst-5-ene-3P, 17a-diol (source No. 15) gave an immediate deep red, changing after 30 minutes to yellow-brown ; androst-5-ene-3/3,17/3-diol (source No. 15) gave no colour.Gestagens-These have twenty-one carbon atoms and are described on p. 566 of steroid^."^ Pregn-4-ene-3,20-dione (progesterone ; source No. 17) and its main reduction products gave pale yellow colours, as did the 3/3,5-en compounds 3P-hydroxypregn-5-en-20-one and pregn- 5-ene-3/3,20P-diol (source No. 15). Corticoids-These have twenty-one carbon atoms and are described on p. 603 of “Steroids.”3 No reaction was obtained with cortisol, cortisone and corticosterone from source No. 17. No samples of corticoids having 3/3-hydroxyl-5-en structure were available for testing. Cardenolides-These have twenty-three carbon atoms and are described on pp. 736, 752 and 754 of v steroid^."^ Those tested were from sources Nos. 5 and 23 and had the 5/3-con- figuration; the positions of the hydroxyl substituents are given after the trivial names, and the colours are those obtained after 30 minutes.Digitoxigenin (3/3,14-dioi), yellow; digoxi- genin (3a,12a,14-triol), pale green; gitoxigenin (3/3,14,16-triol), light yellow ; strophanthidin (3/3,5p,14-triol and CHO group at C,,), yellow, which intensified to about 0.5 C on standing. Conessine-This has twenty-four carbon atoms and is described on p. 857 of steroid^"^; it has the A5 configuration, but a N-dimethyl group at position 3. A sample from source No. 4 gave a negative result. Bile acids-These have twenty-four carbon atoms and are described on p. 422 of steroid^."^ The positions of the substituent hydroxyl groups in the parent cholanic acid are given after each trivial name and are followed by the source number; the colours are those formed after 30 minutes.Lithocholic (3a; No. IS), none; hyodeoxycholic (3a, 6,; No. 1 1), none ; chenodeoxycholic (3a, 7a ; No. 1 1), red-yellow, intensity about 10 ; deoxycholic (3a, 12a; No. ll), yellow-green, intensity about 10; hyocholic (3a, 6a, 7a; No. S), none; acid IT (a-muricholic; 3a, 6P, 7a; No. S), none; acid I (P-muricholic; 3a, Sp, 7p; No. €9, none; acid IV (a-muricholic; 3cc, 6a, 7/3; No. €9, none; cholic (3a, 7a, 12a; No. 16), red-yeilow. Sapogenins (spimfans)-These have twenty-seven carbon atoms and are described on p. 831 of steroid^."^ The main substituents, sources and colours after 30 minutes of the compounds tested were: tigogenin (5a, 3/3-hydroxy ; No. lo), pale yellow; hecogenin (5a, 3/3-hydroxy, 12-0x0; No.lo), pale yellow; 9(11)-dehydrohecogenin (as for hecogenin, but with Ag(11); No. 4), pale yellow; diosgenin, (A5, 3p-hydroxy; No. lo), pale yellow-green; kryptogenin is included in Table I (sample No. 53). CHOLESTERYL ESTERS- The cholesteryl acetate tested was from source No. 6; the others were from source No. 27 (preparation and properties described by Swell and Treadwell12). All the compounds were tested in chloroform; the esters of fatty acids up to C1, were also tested in acetic acid, those with longer chains being insoluble. These tests were carried out by Mr. A. El Sheltawy. Cholesterol in chloroform gives an intensity of 120, compared with 100 in acetic acid. The results are expressed as comparative to the standard in a particular solvent and are given as the cholesterol equivalent in the compound (calculated from the molecular weight).Acetate and propionate, respectively, 60 and 90 (chloroform), 70 and 160 (acetic acid). Dimethylacetate, isobutyrate, n-valerate, isovderate, DL-ethylmethylacetate, isohexanoate, octanoate and decanoate, about 130 (chloroform) and about 90 (acetic acid). Dodecanoate (laurat e) , te tradecanoate (myrist ate), hexadecanoate (palmit ate), oct adecanoate (st earat e) and octadecenoate (oleate) were insoluble in acetic acid and each gave an intensity of about COOK : REACTIONS OF STEROIDS WITH ACETIC ANHYDRIDEJune, 19611 AND SULPHURIC ACID (THE LIEBERMANN - BURCHARD TEST) 379 130 in chloroform. A “fast” reaction, i.e., development of colour after 1-5 minutes, was obtained with most of the esters in chloroform, and the intensity did not decrease greatly after 35 minutes.The benzoate behaved similarly. DISCUSSION OF RESULTS The production of chromogens, some of which are fluorescent, from steroids by relatively large amounts of the so-called Lewis acids, i.e., electron acceptors (including with the con- ventional strong acids such compounds as antimony tri- and pentachlorides) , is a commonly used qualitative and quantitative pr0~edure.l~ The mechanism of the reaction or reactions is obscure,14 and it was not our primary intention to study this, but to obtain information on the value and limitations of the Liebermann - Burchard test. The blue-green colour developed in this test by certain sterols and their derivatives is remarkable for the small amount of sulphuric acid needed, but only slight green-yellow colours are given by androgens, corticoids, bile acids , cardenolides and sapogenins.Oestrogens give, inconsistently, a red- green fluorescence, presumably owing to the phenolic nature of ring A. Trimethyl sterols (lanostane derivatives) give a yellow-green fluorescence. Five broad divisions of colour development and intensity for sterols and their derivatives can be made. These divisions are set out below, the sample numbers referring to Table I. Compounds producing a colour of slight or zero intensity-This class includes 5a- cholestan-3P-01 (sample No. 2) and the corresponding mono- and dimethylstanols (samples Nos. 5 and 6), most oxosteroids (samples Nos. 43, 44, 45 and 46), ‘I-0x0- cholesterol (sample No.52) , 5a-chole~tane-3j3~7P-diol (sample No. 9) and dicholesteryl ether (sample No. 59). The methyl ether (sample No. 58), however, is reactive, possibly because of the relative ease of hydrolysis. Compounds producing a colour intensity half that developed by cholesterol-This class includes coprostanol (sample No. 4) and 24-dehydrocholesterol (sample No. 35). Compounds Producing a colow intensity about the same as that developed by cholesterol- This class has two subdivisions, “fast-acting” and “slow-acting” compounds. The former subdivision includes stenols (samples Nos. 15 and 17) and zymosterol (sample No. 37). The latter subdivision includes all stenols having one double bond in position 5; the substitution of a hydroxy, methyl or ethyl group in the side-chain did not affect the intensity developed (compare results for samples Nos.23, 24, 25, 30, 31 and 32), but the introduction of a double bond other than ethylidene into the side-chain appeared to cause lower intensities (compare samples Nos. 34, 35, 36 and 39, for which the intensities were 70, 50, 70 and 85, respectively). Cholesterol hydrate consistently gave an intensity 10 per cent. greater than that developed by cholesterol. Compo.unds producing a colour intensity about twice that developed by cholesterol- This class has the same subdivisions as Class 3. The “fast-acting” compounds include 4-mOnO- and 4,4-dimethylstenols having double bonds in positions 6, 8, 8(14) or 14 (samples Nos, 19, 20, 21 and 22) and the cholestanediols (samples Nos.7 and 8). The “slow-acting” compounds include epicholesterol (sample No. 12) and thiocholesterol (sample No. 57). Compounds producing a colozlr intensity five to six times that developed by cholesterol- Members of this class are “fast acting,” but the intensities developed decrease after 35 minutes. The compounds all have a double bond in position 7, either alone (samples Nos. 16, 18 and 42) or conjugated with a double bond in position 5 (samples Nos. 33 and 40); others are capable of conversion by dehydration to a conjugated diene (samples Nos. 26, 28 and 29). This “stenadiene” group will form a colour with acetic anyhydride and zinc chloride as dehydrating agent,15 a reaction not given by 7-en stenols; the intensity developed is similar to that de- veloped by cholesterol, and the colour is stable.(This test is useful for differentiating between 7-en and 5,7-dien stenols.) Although oxosteroids are not generally reactive , A7-cholestenone (sample No. 47) is “fast acting” (see also sample No. 50). A few generalisations may be made, namely, that a blue-green colour is obtained only when the steroid has the complete nineteen carbon atoms of the steroid skeleton (for other examples, see paper by Pesez7) and a side-chain of at least eight carbon atoms. The nature380 COOK: REACTIONS OF STEROIDS WITH ACETIC ANHYDRIDE [Vol. 86 and conformation of the substituents at positions 3 and 7 are important. At position 3, the group may be a free or esterified hydroxyl group or a thiol group, and the axial con- formation is apparently more reactive.0x0 sublstituents at position 3 are unreactive, except when there is a double bond at position 7. Cholesteryl esters are of interest in that they are apparently more reactive than the free sterol when in chloroform solution; this established fact16 is still without explanation. At position 7, the presence of a double bond, or the possi- bility of dehydration to form one, confers increased reactivity; the presence of an 0x0 group decreases reactivity . Much of the experimental work was done by Miss D. Young, Miss Marian Greig, Mr. D. I. Cargill, Mr. A. D. Tait and Mr. A. El Sheltawy, to whom I am grateful. Dr. P. Bladon has kindly helped with timely and constructive advice. Part of the expenses was met by a grant from the Scottish Hospital Endowments Research Trust.I thank the persons named in the Appendix for donating the steroids tested. Appendix The steroids tested were obtained from the sources listed below; unless otherwise stated, samples were kindly donated. SOURCES 1. D. H. R. Barton, London. 2. C. A. Baumann, Madison, Wisconsin, 1J.S.A. 3. The late W. Bergmann, Yale University, New Haven, Connecticut, U.S.A. 4. P. Bladon, Royal College of Science and Technology, Glasgow. 5. Burroughs Wellcome & Co. Ltd., London, by courtesy of R. 0. Thomson. 6. This laboratory; most of the compounds were recrystallised or prepared by A. D. Tait. 7. J. W. Cornforth, London. 8. E. A. Doisy, St. Louis University, St. Louis, Missouri, U.S.A. 9. L. F. Fieser, Harvard University, Cambridge, Massachusetts, U.S.A.10. Glaxo Laboratories Ltd., London, by courtesy of T. F. Macrae. 11. G. A. D. Haslewood, London. 12. H. B. Henbest, Queen’s University, Belfast, Northern Ireland. 13. R. N. Jones, Ottawa, Canada. 14. A. A. Kandutsch, Bar Harbor, Maine, U.S.A. 15. W. Klyne, London (Steroid Reference Collection). 16. L. Light & Co. Ltd., Colnbrook, Bucks., by purchase. 17. Merck & Co. Inc., Rahway, New Jersey, U.S.A., by courtesy of M. Tishler. 18. E. Mossetig, Bethesda, Maryland, U.S.A. 19. L. N. Owen, London. 20. Peboc Ltd., London, by purchase. 21. V. Prelog, Zurich, Switzerland. 22. The late 0. Rosenheim, London. 23. Sandoz, Basle, Switzerland, by courtesy of R. A. Ellis. 24. Schering Corporation, Bloomfield, New Jersey, U.S.A., by courtesy of A. Ryer. 25. G. D. Searle & Co., Chicago, Illinois, IJ.S.A., by courtesy of V. A. Drill. 26. R. Stevenson, Brandeis University, Waltham, Massachusetts, U.S.A. 27. C. R. Treadwell, Washington, D.C., U.S.A. 28. W. W. Wells, Pittsburgh, Pennsylvania, U.S.A. 29. U.C.L.A.F., Paris, by courtesy of P. Poirier. REFERENCES 1. 2. 3. 4. 6. 6. 7. Pesez, M., Bull. SOC. Chim. France, 1958, 369. 8. 9. Dam, H., in Cook, R. P., Editor, “Cholesterol,” Academic Press Inc., New York, 1958, p. 5. Moore, P. R., and Baumann, C. A., J . Bid. Chem., 1952, 615, Fieser, L. F., and Fieser, M., “Steroids,” Reinhold Publishing Corporation, New York, 1959. “Handbook for Chemical Society Authors, Reiss, 0. K., Fed. Proc., 1955, 14, 268. Idler, D. R., and Baumann, C. A., J . Bid. Chem., 1953, 203, 389. Neiderheiser, D. H., and Wells, W. W., Arch. Biochem. Bioflhys., 1959, 81, 300. Kandutsch, A. A., and Russell, A. E., J . Biol. Chem., 1960, 235, 2253. Special Publication. No. 14, The Chemical Society, London, p. 132.June, 19611 AND SULPHURIC ACID (THE LIEBERMANN - BURCHARD TEST) 381 Frantz, I. D., jun., Mobberley, M. L., and Schroepfer, G. J., jun., Progress Cardiovascular Diseases, Bladon, P., Cornforth, J . W., and Jaeger, R. H., J . Chem. SOC., 1958, 863. Swell, L., and Treadwell, C. R., J . Biol. Chem., 1955, 212, 141. Kalant, H., Biochem. J., 1958, 69, 79. Bladon, P., iiz Cook, R. P., Editor, 09. cit., p. 84. Meesemaecker, R., and Griffon, H., J . Pharm. Chim., 1930, 11, 572. Kenny, A. P., Biochem. J . , 1952, 52, 611. 10. 11. 12. 13. 14. 15. 16. 1960, 2, 511. Received February 3rd, 1961