Vol. 79 2-METHOXY- AND 2-HYDROXY-OESTRIOL FROM LIVER 361 Layne, D. S. & Marrian, G. F. (1958). Biochem. J. 70, 244. Midgeon, C. J., Wall, P. E. & Bertrand, J. (1959). J. clin. Levitz, M., Spitzer, J. R. & Twombly, G. H. (1958). J. biol. IInvet. 38, 619. Chem. 231, 787. Mueller, G. S. & Rumney, G. (1957). J. Amer. cheem. Soc. Lieberman, S., Tagnon, H. J. & Schulman, P. (1952). J. 79, 1004. din. Inve8t. 3i, 341. Riegel, I. L. & Mueller, G. C. (1954). J. biol. Chem. 210, Loke, K. H. (1958). Ph.D. Thesis: University of Edin- 249. burgh. Ryan, K. J. & Engel, L. L. (1953). Endocrinology, 52, 277. Loke, K. H. & Marrian, G. F. (1958). Biochim. biophy8. Saffran, M. & Jarman, D. F. (1960). Canad. J. Biochem. Acta, 27, 213. Physiol. 38, 303. Loke, K. H., Marrian, G. F., Johnson, W. S., Mayer, W. L. Sandberg, A. A., Slaunwhite, W. R. & Antoniades, H. N. & Cameron, D. D. (1958). Biochim. biophy8. Acta, 28, (1957). Recent Progr. Hormone Re8. 13, 209. 214. Stimmel, B. F. (1959). Fed. Proc. 18, 332. Marrian, G. F., Loke, K. H., Watson, E. J. D. & Panattoni, Szego, C. M. (1953). Endocrinology, 54, 649. M. (1957). Biochem. J. 60, 66. Weil-Malherbe, H. & Bone, A. D. (1957). Biochem. J. 67, Marrian, G. F. & Sneddon, A. (1960). Biochem. J. 74, 430. 65. Biochem. J. (1961) 79, 361 Metabolism of Oestriol in vitro COFACTOR REQUIREMENTS FOR THE FORMATION OF 2-HYDROXYOESTRIOL AND 2-METHOXYOESTRIOL BY R. J. B. KING* Department of Biochemistry, Univer8ity of Edinburgh (Received 2 September 1960) The conversion of oestriol into 2-hydroxyoestriol whereas this vitamin has no effect on the 11pl- and 2-methoxyoestriol by rat- and rabbit-liver hydroxylation of 1l-deoxycorticosterone (Tom- slices described in the preceding paper indicated a kins, Curran & Michael, 1958). new metabolic pathway for oestrogens in vitro. The The O-methylation of catechols is well known results suggested that the oestriol was hydroxyl- (Axelrod & Tomchick, 1958; Pellerin & D'Ionio, ated in the 2 position and this hydroxyl group was 1958), and, like N-methylation (Cantoni, 1951; then methylated to form 2-methoxyoestriol. Cantoni & Vignos, 1954), the methyl group can be A number of papers (reviewed by Grant, 1956a) derived from methionine via S-adenosylmethio- have described the hydroxylation of neutral mine. steroids in vitro, and Mueller & Rumney (1957) This paper describes the cofactor requirements of have studied the 6'oc'-hydroxylation of oestradiol- the rat-liver 2-methoxylating system. 17,B by rat- and mouse-liver microsomal prepara- tions. These hydroxylations all appear to require EXPERIMENTAL molecular oxygen and reduced pyridine nucleotide. The 2-hydroxylation of oestriol represents a major Enzyme preparation8 and methods difference from these reactions in that the hydroxyl The rats were killed by a blow on the back of the neck. group is entering a benzenoid ring structure. Liver and kidney homogenates were prepared by mincing Brodie et at. (1955) have shown that the hydroxyl- the tissue in a Latapie mincer to remove connective tissue ation of aromatic compounds such as acetanilide and then homogenizing with cold 0-25M-sucrose in a glass also requires oxygen and reduced triphosphopyr- homogenizer fitted with a nylon pestle. The ovaries were idine nucleotide, so that some similarity exists be- disrupted in a 0-8 cm. x 15 cm. glass tube with a hand- tween the hydroxylation of aromatic and aliphatic operated metal plunger; usually, 20% (w/v) homogenates compounds. However, Kaufman (1959) has shown were used. The uteri were slit longitudinally and the that a tetrahydrofolic acid derivative is required for inside surface was blotted dry with filter paper. They were the conversion of phenylalanine then cut into small pieces and finely minced with a Mickle into tyrosine, automatic slicer. This brei was added to the appropriate * Present address: John Collins Warren Laboratories of volume of 0 25M-sucrose to give a 20% (w/v) suspension. the Huntington Memorial Hospital of Harvard University When required, the cell debris and nuclei were removed at the Massachusetts General Hospital, Boston, Mass., by centrifuging for 10 min. at 700g at 00 in a MSE refri- U.S.A. gerated centrifuge and the mitochondria by centrifuging 362 R. J. B. KING 1961 for 10 min. at 7000g. The 7000g supernatant fraction was was kindly prepared by Dr E. Brode by the method of usually obtained by centrifuging the whole homogenate for Futterman (1957). Tetrahydrofolic acid was prepared by 10 min. at 7000g. The 105000g supernatant was separated catalytic hydrogenation of folic acid. Adams platinum from the microsomes by centrifuging the 7000g fraction for oxide catalyst (Johnson, Matthey and Co.) (30 mg.) was 30 min. at 105000g in a preparative Spinco model L centri- suspended in 10 ml. of acetic acid and hydrogenated for fuge. The microsomes were suspended in 0-25M-sucrose by 3 hr. at room temperature with gentle shaking. Folic acid gentle homogenization with a glass ball so that 1 ml. of the (100 mg.) was then added and the shaking continued until suspension was equivalent to 1 ml. of homogenate (equiv- the yellow suspension changed to a green solution. The alent to 200 mg. wet wt. of original tissue). catalyst was removed by centrifuging and the tetrahydro- Duplicate incubations were carried out in glass-stoppered folic acid precipitated with 100 ml. of dry ether. The tetra- test tubes and each experiment was repeated at least once. hydrofolic acid was centrifuged off and rapidly washed The extraction and estimation of steroids were carried twice with portions (50 ml.) of dry ether. The whitish pre- out as described in the preceding paper (King, 1961) except cipitate was dried in vacuo and stored under nitrogen in that the alumina-chromatography step was omitted in the the dark at - 17°. experiments in which 2-methoxyoestriol was not estimated. In addition to the steroids mentioned in the preceding By this modified method 80% recoveries of oestriol could paper, the stilboestrol (3:4-di-p-hydroxyphenylhex-3-ene) be obtained regularly. and 17oa-ethynyloestradiol-17, (17oC-ethynyloestra-1:3:5- The Fast Black Salt K (diazotized p-nitrophenylazo- triene-3:17,f-diol) were gifts fromDr G. S. Boyd. The oestriol dimethoxyaniline) derivatives were prepared bythe method was added to the incubation tubes as a solution in pro- of Heftmann (1950). The Girard reaction was carried out as pylene glycol. described in the preceding paper. The sodium borohydride Ether (A.R. grade) was distilled once immediately before reduction was carried out by dissolving the dry material in use. Other solvents and Celite were purified by the methods 1 ml. of methanol and adding 2 mg. of sodium borohydride described by Bauld (1953). (British Drug Houses Ltd.). This solution stood at room temperature for 1 hr. and was then diluted with 10 ml. of 2N-HCI plus 39 ml. of water. The resultant solution was RESULTS extracted three times with portions (50 ml.) of ether and Cofactor requirements for the formation the ether washed twice with 20 ml. of water. The ether of extract was evaporated to dryness under vacuum. 2-methoxyoe8triol by liver homogenate8 Magnesium ions, ATP, L-methionine, DPN and Material8 TPN were required for the optimum conversion of Diphosphopyridine nucleotide (DPN) and barium glucose oestriol into 2-methoxyoestriol. The pyridine nu- 6-phosphate were obtained from C. F. Boehringer und cleotide requirements of this reaction are shown in Sohne (GmbH., Mannheim, Germany). Sodium glucose Table 1; in the absence of DPN and TPN, the 6-phosphate solutions were prepared from the barium salt amount of oestriol metabolized is small and very by precipitating the barium with the calculated amount of little, if any, 2-hydroxyoestriol is produced. sodium sulphate. Calcium lactate was kindly donated by Although the addition of oxidizable substrates Dr J. K. Grant and the sodium salt prepared from this by addition of the calculated amount of sodium oxalate. (lactate and glucose 6-phosphate) has very little Triphosphopyridine nucleotide (TPN), glucose 6-phosphate effect, this is probably due to the presence of endo- dehydrogenase and the sodium salt of adenosine triphos- genous substrates which can reduce DPN and TPN, phate (ATP) were purchased from the Sigma Chemical Co. as the experiment described in Table 7 indicates (St Louis, Mo., U.S.A.). L-Methionlne and folic acid were that the reduced form is required for the 2-hydroxyl- bought from British Drug Houses Ltd. Dihydrofolic acid ation reaction. Table 1. Effect of di- and tri-phosphopyridine nucleotide on metaboliam of oestriol by rat-liver homogenate Figures are given as percentages of oestriol added. All incubations contained 1 ml. of 20 % (w/v) homogenate in 0-25M-sucrose, 150 ,umoles of MgCl2, 5 uLmoles of ATP, 60 ftmoles of L-methionine, 192 ,umoles of potassium phosphate, pH 7-4, 240 ,umoles of KCI and 0-52 ltmole (150 jug.) of oestriol in 0-035 ml. of propylene glycol. Final vol., 3 ml. Incubations were carried out under 02 for 1 hr. at 370. G-6-P, Glucose 6-phosphate. 2-Methoxy- 2-Hydroxy- Oestriol oestriol oestriol Total Additions recovered formed formed recovery None 63 0 1 64 0-7 ,mole of DPN +4-5 ,moles of lactate 51 4 1 56 1-4 ,umoles of DPN +9 umoles of lactate 42 5 1 48 0-7 pDmole of TPN +4-5 !&moles of G-6-P 37 9 5 51 1-4 umoles of TPN +9 jAmoles of G-6-P 7 11 6 24 0-7 Fmole of DPN +0-7 amole of TPN+ 4-5 ,umoles 17 18 6 41 of lactate +4-5 ,umoles of G-6-P 0-7,umole of DPN +0-7,umole of TPN 19 15 5 39 0-7 1Amole of DPN +0-7 pmole of TPN (oestriol 73 0 0 73 added just before extraction) Vol.