Dehydrogenation of sterols by the protozoan Tetrahymena pyriformis.

Abstract Evidence is provided that Tetrahymena pyriformis is capable of converting 5α-cholest-7-en-3β-ol, 5α-cholestan-3β-ol, and cholest-5,24-dien-3β-ol (desmosterol) to products having a 5,7,22-triene system. In the case of desmosterol the C-24 double bond is largely retained. Studies with [6α-3H]- and [6β-3H]5α-cholest-7-en-3β-ol indicate that the introduction of the 5 double bond in these compounds proceeds with the abstraction of the 5α- and 6α-hydrogens. The 6β-tritium atom is completely retained. The introduction of the 5,6 double bond is accompanied by a considerable isotope effect. This is in contrast to the previously reported observations that the introduction of the 7 and 22 double bonds does not involve an isotope effect.

In the case of desmosterol the C-24 double bond is largely retained.
Studies with [SOL-aH]-and [6/3-aH]50L-cholest-7-en-3/3-ol indicate that the introduction of the 5 double bond in these compounds proceeds with the abstraction of the 5~ and 6a-hydrogens. The 6p-tritium atom is completely retained. The introduction of the 5,6 double bond is accompanied by a considerable isotope effect. This is in contrast to the previously reported observations that the introduction of the 7 and 22 double bonds does not involve an isotope effect.

The protozoan
Tetrahymena pyriformis has been shown to convert mevalonic acid (MVA) to tetrahymanol (I) via a nonoxidative proton-initiated cyclization of squalene (l- 4). No evidence has been found for the production of cholesterol or related sterols by this organism or for an absolute sterol nutritional requirement (Reference 5 and references therein).
However, it has been demonstrated that exogenous cholesterol inhibits tetrahymanol production and is itself effic;ently metabolized to the cholesta-5,7,22-triene-30-01 (IIa) by the organism (6)(7)(8)(9)(10). We have proven that the dehydrogenations proceed by the abst,raction of the C-7/3 and C-8fl hydrogens and of the 22.pro-R hydrogen (7,8). Our results were subsequently confirmed by other investigators (9,lO) who also showed that a 23-pro-S proton is removed in the trans AZ2 formation (9). It is worthy of note that the C-7 and C-22 double bond formations proceed with the removal of cis-orientated hydrogens. Several mechanisms were suggested for cis dehydrogenations of sterols (7,8,10,11 and 210 ml of water per 100 g of silica. The plates were dried, activated, and stored in the dark. Solvent systems used were: I, ethyl acetate-hexane (1: 9) ; II, acetone-chloroform (2 : 98). Plates were scanned for radioactivity with a Vanguard model 885 instrument.
[26-WJCholesterol and [26-W]desmosterol were obtained from New England Nuclear and were used without further purification.
Growth of T. pyrijormis and Incorporation of Xterols-The double culture technique was used for incorporation of radioactive substrates (l-3).
A fresh inoculum of T. pyrijormis (100 ml) was prepared by incubation in the normal medium without added glucose at 28-30" for 48 hours. The sterol under investigation (2.5 to 3 mg) was dissolved in ethanol (1 ml) and added to a sterilized l-liter solution of the growth medium in a 2.8-liter Fernbach flask. The medium was then supplemented with 20% glucose solution (50 ml) and 100 ml of the fresh inoculum of T. pyrijormis.
The flask was shaken vigorously at 2830" in the dark for 72 hours. The cultures were cooled to 4" for 2 hours and the cells were harvested by continuous centrifugation.
The wet cells from each flask were saponified with KOH (5 g) in aqueous ethanol (1: 1, 30 ml) at reflux for 2 hours in an atmosphere of nitrogen. The volume of the cooled solution was reduced under vacuum and the nonsaponifiable material was isolated by extracting several times with hexane. The organic phase was washed with saturated NaCl solution and with water before drying (Na$Od) and removal of solvent under vacuum. The sterols were separated and purified as described below, all operations being performed in subdued light.
The solution was cautiously diluted with methanol and the solvents were removed under a stream of nitrogen. The crude product was purified by preparative thin layer chromatography on silica gel in System I and the single unresolved band (2.2 x lo5 dpm) was eluted with ethyl acetate.
The upper band was further purified by thin layer chromatography and after &lution with nonradioactive cholest-7-en-3@-yl acetate was crystallized four times to give a constant specific activity of 8.8 x lo5 dpm of i4C per mmole; 3H:14C ratio 13.6.
The lower band was also purified by rechromatography. Its structure was defined as cholest,a-5,7,22-trien-3@yLacetate (IIb) by its spectral properties (6) (3.5 x lo5 dpm of 14C; 3H:14C ratio 25.7) was diluted with nonradioactive material (6 mg) and incubated with T. pyrijormis as described above. After acetylation and purification by thin layer chromatography the radioactive product (2.7 x lo5 dpm of 14C) was further fractionated on silica gel-silver nitrate (System I) to give two major bands. The upper band was eluted (0.6 x lo5 dpm of i4C) and crystallized with authentic cholest-7-en-3P-yl acetate to a speafic activity of 3.8 x lo5 dpm of 14C per mmole; 3H:14C ratio 60.3.
The lower band was purified by thin layer chromatography as described above. The structure of the compound was confirmed as IIc by ultraviolet and mass spectrometry: X,,, 262, 271, 281.  (2 11) and platinum oxide (50 mg) were added and the mixture was shaken in an atmosphere of hydrogen. The solution was filtered and shaken with NaHCOz solution after which it was washed and dried (Na2S04) . Cholestanol was isolated by preparative thin layer chromatography on silica gel-silver nitrate (System II, developed twice at 4') and elution with ethyl acetate. The product was crystallized twice from ether-methanol to give [26J%]cholestanol (18 mg, m.p. 141-142', specific activity 5.6 x 10' dpm per mmole) ; mass spectrum, m/e 388 (M+, base peak), 373, 355, 331, 262, 257, 234, 233, 217, 215.
Incubation of [.Z6-~4C]cholestanol with T. pyrijormis-The purified [26-W]cholestanol (2.8 mg; 4.1 x lo5 dpm; specific activity 5.6 x 107 dpm per mmole) was added to a growing culture of T. pyrijormis in 1 liter of medium, as described above. The cells were then harvested and saponified, and the sterols were extracted with hexane. The recovered sterols (2.3 X lo5 dpm) were purified by preparative thin layer chromatography (System I), to give a single unresolved radioactive zone, which was fractionated by thin layer chromatography on silica gel-silver 5ar-cholest-7-en-3P-ol (or it,s analogue) (11). The A7 intermediate is dehydrogenated to cholest-5, 'I-dien-3/3-ol and the reaction involves the abstraction of the c&orientated 5aand Ga-hydrogens (12,13). Formally the action of T. pyriformis on ring B of chlolesterol is the reversal of the above process.
In this instance, the C-5 double bond is present and the C-7 double bond is introduced. As mentioned earlier, the introduction of the A7 unsaturation by T. pyrijormis entails the elimination of the &-orientated 7/3-and 8/3-hydrogens.
In view of the opposite sequence of the dehydrogenation steps occurring in the biosynthesis of cholesterol and in the metabolism by T. pyrijormis, we wished to explore the ability of the protozoan to metabolize 5cu-cholest-7-en-3P-01. Consequently, an exploratory incubation of [6-3H2]cholest-7-en-3/3-ol with the organism was carried out. We noted that the protozoan effi- of the A5 double bond involves loss of the cisoriented 5a-and Gar-protons, the 6&proton being retained. Clearly, this is another example of an over-all cis dehydrogenation at nonactivat.ed carbon atoms and follows the same over-all steric course as the 5,6 dehydrogenations involved in the biosynthesis of cholesterol in rat livers and ergosterol in yeast.
Worthy of note is the fact that no isotope effect was observed in the removal of the 7/?-or 22-pro-R-protons of cholesterol by T. pyriformis (7-Q). A similar isotope effect was first observed in the conversion of cholest.-7-en-3/3-ol to cholesta-5,7dienol in a rat liver enzyme system (13).
This effect may reflect a similar enzymic process in these two cases.
We have also incubated They noted that while the presence of C-24 ethylene or trans-C-24(28)-ethylidene moieties do not interfere with the dehydrogenation at C-22, a c&24(28)-ethylidene group prevents the introduction of the C-22 double bond. They concluded that dehydrogenation at C-22 can occur when carbons C-20, 22, 23, 24, 28, and 29, if present, can lie in one plane, the other side of the plane being exposed for the abstraction of k-oriented C-22 and C-23 hydrogens.