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. S, B, and C indicate authentic squalene, brassicasterol and chole sterol, respectivley. 1, 7, 8, 9, 10 and 14 in dicate the fractions obtained by column chro matography. 1201

drica is capable of synthesizing sterols from acetate.

Discussion

It is generally accepted that sterols occur in all aminals, plants, and microorganisms.

As an exceptional case, bacteria and blue-green algae have been conceivable to contain no sterol in the cells. However, SCHUBERT et al. have reported the presence of chole sterol, campesterol, stigmasterol, and ƒÀ-sitosterol in Escherichia coli.15) Also, DE

SOUZA and NES have studied on the isopentenoid constituents of the blue-green alga,

P. luridum, and showed that this alga contained phytol, squalene, and the sterol mixture composed of 24-ethyl-‡™7,22-cholestadienol, 24-ethyl-‡™7-cholestenol, cholesterol, 24- ethyl-‡™5,22-cholestadienol, 24-ethyl-‡™5,7,22-cholestatrienol, and 24-ethyl-‡™5,7-cholestadi enol.5) Moreover, REITZ and HAMILTON6) have demonstrated the presence of cholesterol and ƒÀ-sitosterol in the blue-green algae, A. nidulans and F. diplosiphon, with suggestion that sterols may be bound tightly in the cells of these algae.

In the present study, it was also found that the blue-green alga, A. cylindrica, grown as eptically on the medium containing only the salts and without sterols, contained bras sicasterol and a small amount of 22-dehydrocholesterol, cholesterol, and 22-dihydrobras sicasterol. In addition, the occurrence of sterols in A. cylindrica was further confirmed by the fact that the incubation of the cells of A. cylindrica witn acetate-1-14C gave radio active squalene and sterols. The sterol content of A. cylindrica (0.253mg/g of fresh cells) obtained in the present study was very high as compared with that of P. luridum (0.03mg/g of fresh cells) which was reported by DE SOUZA and NES.5) This may be attributed to the discrepancy of extraction methods for sterols from the cells.

Considering the above-mentioned facts, there is almost no doubt that blue-green algae as well as other algae contain sterols in their cells.

Acknowledgements

The authors wish to express thanks to Prof. K. KASHIWADA, University of Kagoshima, for his continued encouragement in this study. The technical assistance of Miss T. OZEKI is also acknowledged.

References

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