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Bulletinof theJapanese Society of ScientificFisheries 38(10), 1197-1202,(1972) Occurrence of Sterols in the Blue-Green Alga , Anabaena cylindrica Shin-ichi TESHIMA and Akio KANAZAWA* (Received August 26, 1972) The composition and biosynthesis of sterols in the blue-green alga , Anabaena cylindrica, were investigated. 1) From the blue-greeen alga cultivated aseptically in a defined medium, sterols were isolated by the digitonin-precipitation method. 2) The identification of sterols was performed by gas-liquid chromatography and mass spectrometry. 3) The sterols isolated from the blue-green alga were composed of brassicasterol (90%), cholesterol (8%), 22-dihydrobrassicasterol (2%), and 22-dehydrocholesterol (<1%). 4) The incubation of the blue-green alga with acetate-1-14C gave both radioactive squalene and sterols. The results suggest that the enzyme systems for biosynthesis of sterol exist in the blue-green alga under investigation. A majority of works have been carried out on the sterols of marine plants.1) In the red algae, cholesterol and other C27-sterols have been reported to occur as main sterols. The brown algae have been found to contain large amounts of fucosterol and minor other sterols. The green algae and diatoms have been shown to contain principally a variety of C28- and C29-sterols. In the case of the blue-green algae, several attempts to isolate sterols have been unsuccessful.2-4) Accordingly, it has been long recognized that the blue-green algae contain no sterols in the cells as most bacteria. Recently, two groups of workers have demonstrated that the blue-green algae, Phormidium luridum5), Anacystis nidulans6), and Fremyella diplosiphon6), contained sterols in their cells. These findings raised our interest in the occurrence and origin of sterols in the blue-green algae. The present paper deals with the composition and biosynthesis of sterols in Anabaena cylindrica. Materials and Methods A. cylindrica. A. cylindrica was kindly supplied by Professor H. IIZUKA, the In stitute of Applied Microbiology, University of Tokyo. Two loops of the blue-green alga were inoculated in 100ml Erlenmeyer flask containing 50ml of the modified DET MER'S medium (pH 7.6)7) and cultivated aseptically with aeration (flow rate of air, 100- 300ml/min) under the light (2000-4000 Ix) of white fluorescent lamp at 22-25•Ž for 2 weeks. The culture medium contained the following salts per liter: KNO3, 1.0g; CaCl2 * Faculty of Fisheries , University of Kagoshima, 470 Shimoarata-cho, Kagoshima City, Japan (手 島 新 一 ・ 金 沢 昭 夫:鹿児島 大 学 水 産 学 部) (印 刷 費 負 担) 1198 2H2O, 0.01g; MgSO4•E7H2O, 0.25g; K2HPO4, 0.25g; NaCl, 0.1g; FeSO4. 7H2O, 2.0mg; MnSO4•E7H2O, 2.5mg; ZnSO4•E7H2O, 0.222mg; CuSO4•E5H2O, 0.079mg; H3BO4, 2.86mg; Na2MoO4, 0.021mg. The large scale cultivation was carried out in 1-liter Erlenmeyer flask containing 700ml of culture medium in the same manner. After cultivation, the cells were collected by centrifugation (3000 rpm, 10 min) and then washed twice with phosphate buffer (pH 7.4). Isolation of sterols. The cells of A. cylindrica were saponified with ethanol-50% aqueous potassium hydroxide (10:4) at 80•Ž for 17 hr. The unsaponifiable matters were extracted with ether in the usual manner, and then the 3ƒÀ-sterols were isolated from the unsaponifiable matters by the digitonin-precipation method.8) Derivative formation. Steryl acetate was formed by addition of acetic anhydride dry pyridine (1:1) and standing for 48 hours at room temperature. Hydrogenate was obtained by dissolving sterol in ethyl acetate and bubbling with pure hydrogen in the pre sence of acetic acid and platinum oxide. Chromatography and mass spectrometry. Gas-liquid chromatography (GLC) was conducted with a Shimadzu GC-3AF gas-chromatograph by using 1.5% SE-30 and 1.5% OV-17 for columns.9) In thin-layer chromatography (TLC), two types of adsorbent, Kiesel gel G (Merck) and a mixture of Kiesel gel G-silver nitrate (4:1, w/w)10) were used. In column chromatography, alumina (Merck, grade II) was used for preliminary isolation of sterols and hydrocarbons from the unsaponifiable matters. In order to separate the individual sterol components, the steryl acetate mixture was chromato graphed on a silver nitrate-impregnated silicic acid (Mallinckrodt Co., U. S. A.)10) with hexane-benzene. Mass spectrum was measured with the Hitachi RMU-6D instrument (chamber voltage, 70 eV). Incubation of A. cylindrica with acetate-1-14C. The resting cells (15g) grown on the modified DETMER's medium for 9 days were collected and incubated with 50 ƒÊCi of acetate-1-14C (specific activity, 50.0 mCi/m mole in 75ml of phosphate buffer (pH 7.0) containing DL-methionine (10mg), penicillin G (9•~104 units) and aureomycin (2.4mg). The incubation was carried out with shaking at 22•Ž for 21 hr. After incubation, the cells were collected and washed 4 times with distilled water by centrifugation (4000 rpm, 10 min). Incorporation of radioactivity. From the cells of A. cylindrica incubated with acetate- 1-14C, the unsaponifiable matters were isolated and then dissolved in hexane. After ad dition of about each 5 mg of authentic cholesterol and brassicasterol, the hexane-soluble matters were chromatographed on alumina (10g, 1.0•~13.0cm). The elution was carried out stepwise with each 100ml of hexane, hexane-benzene (1:1), benzene, and benzene-ethyl acetate (1:1). The each fraction (25ml) was monitored by TLC and GLC. An aliquot of the radioactive elutates was subjected to TLC on Kiesel gel G 1199 with benzene-ethyl acetate (4:1) and radioautographed by exposing the chromatogram for 2 weeks. In the radioautography , the reference compounds on the plate, cholesterol, brassicasterol, and squalene, were visualized by spraying sulfuric acid-ethanol (1:1) followed by heating at 105•Ž for 5 min. The radioactivity was measured with a Beck man liquid scintillation counter LS-230 by using PPO as a scintillator. Results Sterol composition. The cells (17.0g, wet) of A. cylindrica yielded the unsaponifi able matters (42.0mg) and 3ƒÀ-sterols (4.3mg). The 3ƒÀ-sterols were purified by recrystal lization from methanol. As shown in Table 1, the GLC of the 3ƒÀ-sterols revealed the presence of four components (peaks 1, 2, 3, and 4). The peaks 1, 2, 3, and 4 were Table 1. Sterol composition of A. cylindrica determined by GLC. * 1 .5% SE-30: 200cm•~4mm I. D., temp. 225•Ž 1.5% OV-17: 300cm•~4mm I. D., temp. 243•Ž ** Relative to cholesterol identical with 22-dehydrocholesterol, cholesterol, brassicasterol, and campesterol in the retention times, respectively. Furthermore, the hydrogenate of this sterol mixture gave cholestanol (9%) and C28-stanol (91%). Since the sterols mixed with 24R- and 24S-alkylated sterols have never been found in biological sources, the peak 4 correspond ing to campesterol was conceivable to be 22-dehydrobrassicasterol which has 24S-methyl group at the side-chain as well as the peak 3 corresponding to brassicasterol. In order to separate the individual components, the sterol mixture was acetylated and chromato graphed on a silver nitrate-impregnated silicic acid with hexane-benzene. As a result, the compound corresponding to the peak 3 in GLC was isolated as a pure steryl acetate.•@ The steryl acetate (m.p. 157•Ž) was saponified with 5% alcoholic potassium hydroxide and the free sterol (m.p. 147•Ž) was obtained. The mass spectrum of this steryl acetate gave the prominent ions at m/e 380 (M+-CH3COOH, M+=molecular ion), 365 [M+- (CH3COOH+CH3)], 337 [M+-(CH3COOH+43 (C22-C27))], 255 [M+-(CH3COOH+ R), R=alkyl side chain], 253 [M+-(CH3COOH+R+2H)], 228 [M+-(CH3COOH+R+ 27)], 213 [M+-(CH3COOH+R+42)], and 211 [M+-(CH3COOH+R+42+2H)]. The lack of the molecular ion peak (M+) at m/e 440 rejected the possibility that this steryl 1200 acetate may be ‡™7-isomer of brassicasteryl acetate.11) The presence of peak at m/e 337, corresponding to the loss of terminal isopropyl group of side chain and acetic acid from the molecular ion, was characteristic of ‡™5,22-steryl acetate.12-14) Moreover, the mass spectrum of free sterol gave the molecular ion peak (M+) at m/e 398 and other prominent peaks at m/e 383 (M+-CH,), 380 (M+-HOH), 365 [M+-(CH3+HOH)], 355 [M+-43 (C2g-C27)], 337 [M+-(43+HOH)], 300 [M+-(97+1H)], 273 (M+-R, R=alkyl side chain), 271 [M+-(R+2H)], 255 [M+-(R+HOH)], 253 [M+-(R+2H+HOH)], 231 (M+-(R+42)] and 213 [M+-(R+42+HOH)]. These data supported that the com pound corresponding to the peak 3 was brassicasterol. From the above results, it was concluded that A. cylindrica contained 22-dehydro- cholesterol (<1%), cholesterol (8%), brassicasterol (90%), and 22-dihydrobrassicasterol (2%). Sterol biosynthesis. For the purpose of obtaining the decisive evidence of the occurrence of sterols in A. cylindrica, the biosynthesis of sterols from acetate was in vestigated by using tracer technique. The unsaponifiable matters (40mg) of the A. cylindrica incubated with acetate-1-14C gave high radioactivity (500,000 dpm). As shown in Fig. 1, the column chromatography of unsaponifiable matters showed the presence of radioactive hydrocarbons, sterols, and unknown compound. As shown in Fig. 2, the TLC of the fractions obtained by column chromatography revealed that acetate-1-14C was incorporated into both squalene and sterols. These results indicated that A. cylin Fig. 1. Column chromatography on alumina of Fig. 2. Radioautogram of the fractions obtained the unsaponifiable matters of the A. cylindrica by column chromatography on alumina of the incubated with acetate-1-14C. unsaponifiable matters. I, hydrocarbons; II, sterols; The each fraction was subjected to TLC on III, unknown compound Kiesel gel G with benzene-ethyl acetate (4:1) and radioautographed.

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