Occurrence of Plant Sterols in Aquatic Vertebrates TORU TAKAGI, AKIRA SAKAI, KENJI HAYASHI, and YUTAKA ITABASHI, Department of Chemistry, Faculty of Fisheries, Hokkaido University, Hakodate~ Japan

ABSTRACT

plant sterols were found by gas fiquid chromatography in the sterols of five species of aquatic vertebrates; mackerel (Scomber japonicus), rainbow trout (Salmo gairdnerii), smelt (Osmerus dentex), sardine (Sardinops melanosticta) and chimera (Chimera phantasmal The sterols of chimera liver, sardine flesh and sardine viscera contained 9.0, 2.4 and 3.1% of C28 and C29 sterols in addition to 86.7, 96.6 and 95.2% of cholesterol. The occurrence of norcholestandienol, campesterol, #-sitosterol and C28 stanol was shown by combined gas chromatography-mass spectrometry. Sperm whale (Physeter catodon) sterols consisted of more than 99% cholesterol with only traces of 22-dehydro- cholesterol.

INTRODUCTION

It was once accepted that higher animals and most of lower animals contained cholesterol with minor components of C27 derivatives, e.g., c h o l e s t a n o l , 7-dehydrocholesterol and 7- cholestenol, while plants and some of lower animals contained a variety of C 26-C29 sterols (1). Recently, plant sterols were found in man and other higher animals as minor components, and their physiologically important roles are now recognized (2-6).

It is well known that the main sterol of fish is cholesterol. However, minor sterols in fish have not been reported. This study was carried out to investigate the minor components in fish sterols, and we now report the occurrence of C28 and C29 plant sterols in fish.

MATE RIALS AND METHODS

Commercial samples of the crude bead and body oils from a sperm whale captured in the Antarctic Ocean were obtained. The head oil was used in the gas liquid chromatographic (GLC) analysis of the wax esters (7). The flesh samples of mackerel (Scomber japonicus), sa rd ine (Sardinops melanosticta), rainbow trout (Salmo gairdnerii), and smelt (Osmerus dentex) were obtained commercially at Hako- date in November and December 1977. A chimera (Chimera phantasma) captured in the deep sea off New Zealand was obtained from a fishery company.

Extractions of tipids were carried out by the Bligh and Dyer method (8). The unsaponi- flables were obtained by ether extraction of the saponified lipids in the ordinary way. The sterol fraction was separated by preparative thin layer chromatography using Merck Kiesel Gel G (Type 60) plates and n-hexane-ether-acetic acid (60:40:1) for development.

GLC analyses of sterols were carried out with a Shimadzu gas chromatograph Model

6AM equipped with dual hydrogen flame detectors on 1 m x 3 mm ID and 2 m x 3 mm ID glass columns packed with 2% OV-1 on 80-100 mesh Chromosorb W AW DMCS and 5% SE-30 on Chromosorb W 100-120 mesh, respec- tively. The carrier gas was nitrogen.

Gas chromatography-mass spectrometry (GC-MS) analyses of sterols were carried out with a Hitachi E1 instrument coupled to a com- puter. The GLC separations were performed on 1 m x 3 mm ID glass columns packed with 2% OV-1 on 80-100 mesh Chromosorb W. The carrier gas was helium, and the column tem- perature was programmed from 190 to 280 C at a rate of 5 C/min. The injector, separator, and ion source were kept at 290, 300 and 180 C, respectively. The spectra were taken at an ionizing voltage of 20 eV and accelerating vol- tage of 3.2 kV. Mass chromatograms and bar- graphs were constructed by the computer.

Authentic samples of/3-sitosterol, cholesterol and campesterol were obtained from Wako Pure Chemical Industries Ltd., Osaka, Japan. Cho- l e s t e r o l containing norcholestandienol, 24- methylenecholesterol and desmosterol (obtained from a crab, Erimacrus isenbeekii, cf. Yasuda (9)) was also used as a reference standard.

RESULTS AND DISCUSSION

T h e t y p i c a l gas chromatogram of the chimera liver sterols is shown in Figure 1. The retention times of the peaks 1, 3, 4, 5 and 7 were in agreement with those of norcholestan- d i e n o l , cholesterol, 24-methylenecholesterol, campesterol and fl-sitosterol, respectively. The sterol compositions of chimera liver, sardine viscera and sardine flesh are shown in Table I as peak area percents. It is noteworthy that they contain significant amounts of plant sterols. In particular, the plant sterols in the chimera liver reach almost 10%. Such high percentages of plant sterols in vertebrate sterols have not

TORU TAKAGI, AKIRA SAKAI, KENJI

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FIG. 1. Gas chromatogram of chimera liver sterols. Hitachi GC-MS El, 1 m • 3 mm ID glass column packed with 2% OV-1 on 80-100 mesh Chromosorb W. Detector: Total ion collector. Column temperature was programmed from 190 to 280 C at 5 C/min.

HAYASHI, AND YUTAKA ITABASHI

h i the r to been repor ted . In the o the r fish, the m i n o r s terol c o m p o n e n t s wh ich exceed 0.1% were 2 2 - d e h y d r o c h o l e s t e r o l and campes te ro l u n d e r the c ond i t i ons of this e x p e r i m e n t , and the choles te ro l c o n t e n t s were more t ha n 98%. The sterols of spe rm whale head and b o d y oils con ta ined more t h a n 99% of cholesterol , and C28 and C29 sterols were n o t f ound in t he m. Thei r s terol c o m p o s i t i o n s are s h o w n in Table II.

The typical GC-MS s p e c t r u m of the p l an t sterols f r o m the ch imera liver is s h o w n in Figure 2. GLC peaks 1-8 s h o w e d the fol lowing

ma jo r molecu la r ions: 370, no rcho le s t and ieno l , for peak 1; 384, 22 -dehydrocho le s t e ro l , for peak 2; 386, choles terol , for peak 3; 398, 2 4 - m e t h y l e n e c h o l e s t e r o l , for peak 4; 400, campes te ro l , fo r peak 5; 402, C28 s tanol , for peak 6; 414, /~-sitosterol, for peak 7; and 400, C28 m o n o u n s a t u r a t e d sterol , for peak 8. T h e mass s p e c t r o m e t r y (MS) pa t t e rn s were com- pared wi th those descr ibed in the l i tera ture (10).

The molecular ion peaks were the base peaks as expec ted f rom the mild opera t ing condi t ions

TABLE I

Compositions of Sterols in Chimera Liver, Sardine Viscera and Flesh (GLC Peak Area %)

Peak Chimera Sardine number Sterol liver Viscera Flesh

1 Norcholestandienol 0.6 0.2 T a 2 22-dehydrocholesterol b 3.7 1.5 1.0 3 Cholesterol c 86.7 95.2 96.6 4 24-methylenecholesterol 2.5 T --- 5 Campesterol 2.1 2.6 2.1 6 C28 stanol d 1.5 0.4 0.3 7 fl-sitosterol 2.5 0.1 T 8 C28 sterol d 0.4 T T

aTrace, less than 0.1%. bpresumed by its retention time and GC-MS data. CGC-MS data suggest the overlapping of cholestanol peak. dpresumed by GC-MS data.

TABLE ti

Compositions of Sterols in Some Aquatic Vertebrates (GLC Peak Area %)

Animal Part 22-dehydrocholest er ol Cholesterol Campesterol

Mackerel White muscle 0.8 98.9 0.3 Dark muscle 1.3 98.2 0.5 Viscera 0.7 99.2 0.1 Head T 99.8 0.2

Rainbow trout Whole --- 99.7 0.3 Sperm Whale Body 0.5 99.5 ---

Head 0.8 99.2 --- Smelt Whole --- 98.8 1.2

LIPIDS, VOL. 14, NO. 1

PLANT STEROLS IN AQUATIC VERTEBRATES 7

100 414

M

231 213 229

:.. 1 . . h, 200

303

246 275

,h,. h hill. , , . ..,,.,, 300

m/e

396

399

,.b!, [ ,,1, 400

FIG. 2. GC-MS spectrum of GLC peak 7 (#-sitosterol) of chimera liver sterols. Operating conditions as given in text.

for the ionization; the low ionizing voltage (20 eV) and the low temperature of the ion source (180 C) reduce fragmentation. Exceptionally, the molecular ion peak was not the base peak in the spectra for the GLC peak 3, but it was one of the main peaks. The peak of m/e 388 accompanied by the molecular ion peak of cholesterol at m/e 386 suggests overlapping of the cholestanol peak with peak 3 in the gas chromatogram. The fragment ion peaks M+-I 5 (CH3) , M+-18 (H20) , M+-15-18 followed the molecular ion peaks as an ordinary pattern in MS. The ion peaks m/e 271 (M+-R-2H, R = side chain) and adjacent peaks m/e 273 (M+-R) were common in the spectra for all GLC peaks. They indicated the presence of the monounsaturated sterol ring. These facts and the ion peak 400 accompanied by the molecular ion peak 402 for the GLC peak 6 suggested the tailing of campesterol peak into peak 6.

The presence of an unsaturated CTH ] a and CsH15 side chains in the peak 1 and 2 sterols was supported by their intense fragment ions at m/e 97 and 111 (11). The location of a double bond at 22:23 in the side chain was supported by the intense fragment ion at m/e 300, which was derived from the allylic cleavage with one hydrogen transfer (12).

Mass chromatograms of the chimera liver sterols are shown in Figure 3. The presence of peaks 1-7 is in harmony with the interpretation for the peak components given by the GLC retention times. Similar results were obtained with sterols of sardine flesh and viscera. The expected m/e fragment ions were detected

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TIC

! 414 x 5

402 x 5

400 x 5

398 x 5

386 x.1

384 x 2

1 370 x 5

SCAN

FIG. 3. Mass chromatograms of chimera liver sterols. TIC: Chromatogram by a total ion collector. Relations between peak numbers and components are shown in Table I.

along with those of the respective molecular ions in the same scans.

The results presented here clearly establish that some kinds of fish contain small amounts of plant sterols with a major component cho- lesterol. Further work is proceeding in our laboratory to determine the origin and the effect of plant sterols in fish.

REFERENCES

1. Fieser, L.F., and M. Fieser, "Steroids," Reinhold

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TORU TAKAGI, AKIRA SAKAI, KENJI HAYASHI, AND YUTAKA ITABASHI

Publ. Co., New York, 1959. 2. Kuksis, A., L. Marai, J.J. Myher, and K. Gehr,

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Physiol. 57B:37 (1977).

8. Bligh, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 37:911 (1959).

9. Yasuda, S., Comp. Biochem. Physiol. 44B:41 (1973).

10. Knights, B.A., J. Gas Chromatogr. 5:273 (1967). 11. Id le r , D.R., P.M. Wiseman, and L.M. Safe,

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(1968).

[ R e c e i v e d A u g u s t 28 , 1978 ]

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