SHORT COMMUNICATIONS i n s t e a d in t h e family T e t t i g o n i d a e , w h i c h includes the longhorned grasshoppers and k a t y d i d s . Results p r e s e n t e d in this p a p e r s h o w t h a t 2 - m e t h y l a l k a n e s c h a r a c t e r i s t i c o f crickets are a b s e n t , w h e r e a s c o n s i d e r a b l e a m o u n t s of 3-methyl, internally branched monomethyl-, a n d d i m e t h y l a l k a n e s are p r e s e n t . T h e alkanes o f A. simplex are similar to t h o s e of t h e grassh o p p e r s Melanoplus sanguinipes a n d Melanoplus packardii in t h a t t h e n-alkanes c o n t a i n t h e s h o r t e s t c h a i n l e n g t h c o m p o n e n t s (C23 t o C 3 3 ) , t h e 3 - m e t h y l a l k a n e s are f r o m C28 t o C32, a n d t h e i n t e r n a l l y b r a n c h e d m o n o m e t h y l alkanes a n d d i m e t h y l a l k a n e s c o n t a i n t h e longest c h a i n c o m p o n e n t s . N o t e n o u g h insects have b e e n s t u d i e d t o date t o p r o p o s e c h e m o t a x o n o m i c rules, y e t t h e i n t e r e s t i n g p a t t e r n s of m e t h y l b r a n c h e d h y d r o c a r b o n s n o t e d a b o v e in f o u r families o f O r t h o p tera suggest t h a t s u c h a possibility m a y exist. L A R R Y L. J A C K S O N D e p a r t m e n t of C h e m i s t r y Montana State University Bozeman, Montana 59715 G A R Y L. B L O M Q U I S T D e p a r t m e n t of C h e m i s t r y U n i v e r s i t y o f S o u t h e r n Mississippi H a t t i e s b u r g , Mississippi 3 9 4 0 1
79 ACKNOWLEDGMENTS
This work was inspired by G.L. Baker. John E. Henry, USDA, Range Insect Laboratory, Bozeman, MT, provided the insect collection.
REFERENCES 1. Baker, G., J.H. Pepper, L.H. Johnson, and E. Hastings, J. Insect Physiol. 5:47 (1960). 2. Soliday, C.L., G.J. Blomquist, and L.L. Jackson, J. Lipid Res. 15:399 (1974). 3. Nelson, D.R., and D.R. Sukkestad, Biochemistry 9:4601 (1970). 4. Nelson, D.R., D.R. Sukkestad, and R.G. Zaylskie, J. Lipid Res. 13:413 (1972). 5. Borror, D.J., and D.M. DeLong, "An Introduction to the Study of Insects," Holt, Rinehart, and Winston, New York, NY, 1971, p. 122. 6. Tartivita, ICA., and L.L. Jackson, Lipids 5:35 (1970). 7. Jackson, L.L., Ibid. 5:38 (1970). 8. Jackson, L.L., Comp. Biochem. Physiol. 41B:331 (1972). 9. Nelson, D.R., and D.R. Sukkestad, J. Lipid Res. 16:12 (1975). 10. Hutchins, R.F.N., and M.M. Martin, Lipids 3:250 (1968). 11. Blomquist, G.J., T. Blailock, R.W. Scheetz, and L.L. Jackson, Comp. Biochem. Physiol. (In press). [ R e c e i v e d S e p t e m b e r 29, 1975 ]
In Vitro Activity of the Fatty Acyl Desaturases of Human Cancerous and Noncancerous Tissues ABSTRACT
T h e m i c r o s o m a l d e s a t u r a s e a c t i v i t y of human cancerous and noncancerous t i s s u e s was m e a s u r e d i n v i t r o using 1-14C,- 1 1 , 1 4 - e i c o s a d i e n o i c a n d 1-14C oleic acids as substrates. Tissues used were a case o f ovarian cancer, a u r i n a r y b l a d d e r cancer, a rectal cancer, a n d a n o n specific c o l o n i c ulcer w i t h a p p r o p r i a t e l y n o r m a l tissues. W h e n 1 1 , 1 4 - 2 0 : 2 was used as s u b s t r a t e , r a d i o a c t i v e t e t r a e n e a n d t r i e n e were p r o d u c e d . T h e t e t r a e n e was i d e n t i f i e d b y r a d i o gas c h r o m a t o g r a p h y as a r a c h i d o n i c acid ( 5 , 8 , 1 1 , 1 4 - 2 0 : 4 ) , a n d the triene had a retention time of 5 , 1 1 , 1 4 - 2 0 : 3 . T h u s , t h e possibility arises t h a t a A8 desaturase was involved. I n t h e A 6 desaturase, w i t h t h e u r i n a r y b l a d d e r
c a n c e r o u s tissue, t h e d e s a t u r a s e a c t i v i t y a p p e a r e d t o be d e c r e a s e d in c o m p a r i s o n t o n e i g h b o r i n g tissue, w h e r e a s w i t h t h e c o l o n i c c a n c e r tissue, t h e desaturase appeared t o b e relatively increased, t h o u g h t h e n u m b e r of samples was ina d e q u a t e for c o n f i d e n c e . INTRODUCTION
T h e a t t a c k o n c a n c e r c a n b e carried o u t o n l y w i t h t h o r o u g h k n o w l e d g e of its m e t a b o l i c characteristics. One of t h e p r o p e r t i e s o f r a p i d l y dividing ceils is t h e n e e d for u n s a t u r a t e d f a t t y acids as c o m p o n e n t s of t h e p h o s p h o l i p i d s n e c e s s a r y for t h e f o r m a t i o n o f cell m e m b r a n e s . T h e s o u r c e o f t h e s e f a t t y acids a n d t h e i r m e t a b o l i s m w i t h i n t h e m a l i g n a n t cell t h u s becomes a matter of considerable importance. LIPIDS, VOL. 11, NO. 1
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In particular, the various abilities of the malig- N NH4OH and 0.15 ml 1% Triton) in a nant cells to transform the precursor fatty total volume of 4 ml 0.15 M KC1 and 0 . 2 5 M a c i d s - oleic, linoleic, and linolenic - into the sucrose. The final pH of the solution was 7.1. 609, 6o6, and 603 polyunsaturated fatty acids The vials were shaken for 30 min at 37 C, and apparently required by membrane phospho- the reaction was stopped by addition of 0.1 ml lipids may represent a difference from normal of 9N H 2 SO4. The total lipid was extracted by cells. Chiappe et al. (1) have reported that both the method of Bligh and Dyer (4). After the A9 and A6 desaturases are depressed in two evaporation of the chloroform at 40 C under rat hepatomas as compared with normal liver N2, the lipids were subjected to methanolysis tissue. Because the A5 desaturase may be under with 2 ml of 5% methanolic HC1 (for up to d i f f e r e n t regulation and because different 50 mg) for 30 min at 85 C. The methyl esters tumors in different species may have distinct were extracted with pentane, washed with characteristics, the various desaturases were water, and freed from solvent. One portion of investigated in several human cancers. the methyl ester mixture was separated on t 0% AgNO3-impregnated silica gel plates (5) using 5% acetone in toluene as developing solution. MATERIALS AND METHODS Areas containing fatty acids with different 1 _1 4C_ 11,14-eicosadienoic acid was pur- degrees of unsaturation were located by spraychased from DHOM Products, Ltd. (North ing with an ethanolic solution of 2 , 7 -dichloroHollywood, CA) and 1-14C-oleic acid from fluorescein and by comparison with authentic New England Nuclear Corp. (Boston, MA). standards (Nu-Chek Prep, Inc., Elysian, MN). Both substances were over 99% pure by thin Each fraction was scraped off the plate, and the layer chromatography (TLC) and radio gas radioactivity in each area was measured in the liquid chromatography (GLC) and did not con- l i q u i d s c i n t i l l a t i o n spectrometer (Packard, tain any of the metabolic products concerned. Model 3003). Tissues studied in this research were a case A second portion of the methyl ester mixof chronic nonspecific ulcer (21 year old male), ture was separated by TLC as described above, a case of ovarian cancer (51 year old female), a and each fraction was further analyzed by radio case of urinary bladder cancer (61 year old gas chromatography using the Packard gas female), and a case of rectal cancer (75 year old chromatograph with gas proportional counter male). model 894, fitted with a 6 f t x 4 mm glass Tissues were collected during surgical pro- column packed with Apolar 10C (Applied cedures under sterile conditions at the City of Science Laboratory, State College, PA) and Hope Medical Center (Duarte, CA) and were operated at 185 C column temperature. Peaks placed immediately in ice cold Hank's solution. were identified, where possible, by comparison A portion of each specimen was subjected to with authentic standards. histological examination to confirm the malignant or normal condition of the tissues. All RESULTS procedures were carried out at 4 C unless stated otherwise. With 20:2606 as substrate, radioactivity was Each tissue was homogenized with 4 parts found in the areas of the TLC plate corre(v/w) of homogenizing buffer (2), and the sponding to diene, triene, and tetraene (Table homogenate was centrifuged at 600 x g for 30 I). The tetraenoic acid was identified as arachim i n . T h e supernatant was centrifuged at donic (5,8,11,14-20:4) by comparison of its 9,000 x g for 30 min, and the resulting super- GLC retention time with that of an authentic natant was centrifuged at 105,000 g for 90 min. standard. The retention time of the single comThe pellet from the last centrifugation, the ponent of the triene zone was the same as that microsomal fraction, was suspended in homog- of 5,11,14-20:3. Ozonolysis was carried out enizing buffer (2:1, v/v) with sonication, and with both fractions (6) but, because of the the protein content was measured by the small amounts involved, was not definitive. method of Lowry et al. (3). Including the buf- Throughout the experiment, the total radiofer solution, each vial contained 3-10 mg of activity of tetraene was higher than that of microsomaI protein and the following addi- triene. tions, in /amoles: adenosine triphosphate, 8; In Table II can be seen the results of expericoenzyme A, 0.4; reduced nicotinamide ade- ments designed to test the activity of the A6 nine dinucleotide, 5; KF, 6; nicotinamide, 2; desaturase. In the urinary bladder cancer, MgC12, 7.5; glutathione, 2.2: phosphate enzyme activity, as measured by diene producbuffer, pH 7.3, 62; and 1 pc (1 pm) of tion per mg microsomal protein, appeared to be substrate fatty acid (in solution in 0.05 ml 0.1 decreased as compared with that of normal t
LIPIDS, VOL. 11, NO. 1
t
.
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SHORT COMMUNICATIONS TABLE I Products Formed from 1-14C-11,14-Eicosadienoic Acid on Incubation with Microsomal Fractions from Human Cancerous and Normal Tissuesa Radioactive Fatty Acids Isolated (%) Tissue
11,14-20:2
20:3
Normal colonic
83
Ovarian cancer
92
3
Urinary bladder cancer
90
4
Urinary bladder normal
92
Colonic
83
5,8,11,14-20:4
7
10 1.9 b
2.7 b 5
0.7 b
1.1 b 6
0.2 b
0.4 b
1.1 b
1.8 b
3
6
11 1.8 b
cancer
Normal colonic
87
3.1 b
6
7 0.7 b
0.7 b
aEach vial contained, in 4 ml, 3-10 mg microsomal protein and the following additions, in /zmoles: adenosine triphosphate, 8; coenzyme A, 0.4; reduced nicotinamide adenine dinucleotide, 5; potassium fluoride, 6; nicotinamide, 2; MgC12, 7.5; glutathione, 2.2; phosphate buffer, pH 7.3, 62; and 1 /~c (1 /am) of substrate fatty acid (in solution in 0.05 ml 0.1 N NH4OH and 0.15 ml 1% triton). Vials were incubated 30 min at 37C, and fatty acid methyl esters were prepared and quantitated using radio gas chromatography. bExpressed as percent of radioactive fatty acids produced per mg of microsomal protein. s u r r o u n d i n g tissue. In t h e c o l o n i c c a n c e r , h o w ever, t h e A 6 d e s a t u r a s e a c t i v i t y a p p e a r e d to be increased.
TABLE 11 Products Formed from 1-14C-Oleic Acid on Incubation with Microsomal Fractions from Human Cancerous and Normal Tissuesa
DISCUSSION Following i n c u b a t i o n of 11,l 4-eicosadienoic acid w i t h t h e m i c r o s o m a l f r a c t i o n f r o m h u m a n c a n c e r o u s a n d n o n c a n c e r o u s t i s s u e s , radioa c t i v i t y was f o u n d in diene, t r i e n e , a n d t e t r a e n e f a t t y acids. T h e m e t h y l e i c o s a t e t r a e n o a t e h a d t h e s a m e G L C r e t e n t i o n t i m e as m e t h y l arachid o n a t e a n d was p r o b a b l y i d e n t i c a l w i t h t h a t acid. T h e m e t h y l ester of t h e t r i e n o i c f a t t y acid h a d a r e t e n t i o n t i m e c o r r e s p o n d i n g t o t h a t of 5,1 1,1 4-20: 3, t h e e x p e c t e d p r o d u c t f r o m a liver s y s t e m (7), b u t c o u l d n o t be f u r t h e r i d e n t i f i e d b e c a u s e o f t h e small a m o u n t of p r o d u c t available. T h e diene z o n e y i e l d e d o n l y t h e s t a r t i n g s u b s t r a t e , 20: 2606, a n d t h e 1-14C-1 1,1 4-eicosad i e n o i c acid u s e d in t h e e x p e r i m e n t was s h o w n t o be p u r e b y radio G L C a n d n o t to c o n t a i n a n y r a d i o a c t i v e 18: 2. T h u s , t h e r e is a n e e d t o e x p l a i n t h e origin o f t h e a r a c h i d o n a t e . In t h e i n c u b a t i o n s y s t e m , K F was u s e d t o i n h i b i t c h a i n e l o n g a t i o n , a n d r e t r o c o n v e r s i o n o f t h e 1 1 , 1 4 - 2 0 : 2 t o 9 , 1 2 - 1 8 : 2 followed by elongation and desaturation to a r a c h i d o n a t e (8) was s h o w n t o be u n l i k e l y b e c a u s e a trial e x p e r i m e n t u s i n g 1 - 1 4 C - 1 8 : 2 did n o t yield a r a c h i d o n a t e u n d e r t h e s e c o n d i t i o n s .
Radioactive Fatty Acids Isolated (%) Tissue
9-18:1"
6,9-18:2
Urinary bladder cancer
97.5
Urinary bladder normal
99
Colonic cancer
98.8
1.2
Normal colonic
98.8
1.2
2.5 0.14 b 1.0 0.38 b
0.34 b 0.14 b
aEach vial contained, in 4 ml, 3-10 mg microsomal protein and the following additions, in ~zmoles: adenosine triphosphate, 8; coenzyme A, 0.4; reduced nicotinamide adenine dinucleotide, 5; potassium fluoride, 6; nicotinamide, 2; MgC12, 7.5; glutathione, 2.2; phosphate buffer, pH 7.3, 62; and 1 /~c (1 #m) of substrate fatty acid (in solution in 0.05 ml 0.1 N NH4OH and 0.15 ml 1% triton). Vials were incubated 30 min at 37C, and fatty acid methyl esters were prepared and quantitated using radio gas chromatography. bExpressed as percent of radioactive f a t t y acids produced per mg of microsomal protein. T h e a l t e r n a t i v e p a t h w a y s are (a) c o n v e r s i o n o f 11,14-20:2 to 8,11,14-20:3, which has been s h o w n t o p r o c e e d readily t o a r a c h i d o n a t e , a n d LIPIDS, VOL. 11, NO. 1
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(b) c o n v e r s i o n of 1 1 , 1 4 - 2 0 : 2 t o 5 , 1 1 , 1 4 - 2 0 : 3 a n d c o n v e r s i o n of t h i s acid t o a r a c h i d o n a t e , as has b e e n suggested b y Takagi (9) f r o m in vivo e x p e r i m e n t s w i t h rats b u t d e n i e d b y S c h l e n k et
al.
(10).
Recently, S p r e c h e r a n d Lee (7) have carried o u t e x p e r i m e n t s with rat liver m i c r o s o m e s t h a t a p p e a r t o d e n y t h e e x i s t e n c e o f a A 8 desaturase a n d t h e i r results have b e e n c o n f i r m e d in this l a b o r a t o r y . In o u r e x p e r i m e n t s w i t h h u m a n tissues, h o w e v e r , m o r e t h a n 10% of s u b s t r a t e 1 1 , 1 4 - 2 0 : 2 was c o n v e r t e d t o a r a c h i d o n a t e in s o m e cases, a n d e i t h e r of t h e possible r o u t e s w o u l d r e q u i r e d e s a t u r a t i o n at t h e 8 p o s i t i o n . As N a k a z a w a et al. have r e p o r t e d previously ( 1 1 ) , the p h o s p h o l i p i d a n d triglyceride f a t t y a c i d c o m p o s i t i o n o f b i o p s i e d h u m a n liver tissues were distinctly d i f f e r e n t f r o m t h o s e o f c o r r e s p o n d i n g rat tissues, a n d it is possible to c o n s i d e r t h e e x i s t e n c e of a A8 desaturase in h u m a n tissue. F u r t h e r s t u d y will b e n e c e s s a r y t o clarify t h e r o u t e f r o m 20:2006 t o arachid o n a t e a n d t h e co6 p a t h w a y in h u m a n cancerous a n d n o r m a l tissues.
ICHIRO NAKAZAWA T h i r d D e p a r t m e n t of I n t e r n a l M e d i c i n e School o f Medicine Tohoku University Sendal, J a p a n J A M E S F. M E A D D e p a r t m e n t o f Biological C h e m i s t r y a n d L a b o r a t o r y of N u c l e a r M e d i c i n e a n d R a d i a t i o n Biology University o f California Los Angeles, California 9 0 0 2 4
LIPIDS, VOL. 11, NO. 1
R O B E R T H. Y O N E M O T O D e p a r t m e n t of O n c o l o g i c a l Surgery City of H o p e Medical C e n t e r D u a r t e , California 9 1 0 1 0 ACKNOWLEDGMENTS These studies were supported in part by Contract E(O4-1)GEN-12 between ERDA (author, please spell out) and the University of California; by US Public H e a l t h Service R e s e a r c h C a r e e r Award No. GM-K-6-19,177 from the Division of General Medical Sciences, National Institutes of Health; by a grant from the Chancellor's Research Opportunity Fund, UCLA; and by funds from the UCLA General Support Grant. G.A. Dhopeshwarkar and C. Subramanian of this Laboratory provided advice and aid in this research. REFERENCES 1. Chiappe, L.E., M.E. De Tomas, and O. Mercuri, Lipids 9:489 (1974). 2. Brenner, R.R., and R.O. Peluffo, J. Biol. Chem. 241:5213 (1966). 3. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, Ibid. 193:265 (1951). 4. Bligh, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 37:911 (1959). 5. Applied Science Gas Chrom. News Letter, Vol. 13, No. 1, Jan/Feb (1972). 6. Stein, R.A., and N. Nicolaides, J. Lipid Res. 3:476 (1962). 7. Sprecher, H., and C. Lee, Biochim. Biophys. Acta 388:113 (1975). 8. Stearns, E.M., Jr., J.A. Rysavy, and O.S. Privett, J. Nutr. 93:485 (1967). 9. Takagi, T., Bull. Chem. Soc. Jap. 38:2055 (1965). 10. Schlenk, H., D.M. Sand, and J.L. Gellerman, Lipids 5:575 (1970). 11. Nakazawa, I., and S. Yamagata, Tohoku J. Exp. Med., 103:129 (1971). [ R e c e i v e d S e p t e m b e r 15, 1 9 7 5 ]
79 ACKNOWLEDGMENTS
This work was inspired by G.L. Baker. John E. Henry, USDA, Range Insect Laboratory, Bozeman, MT, provided the insect collection.
REFERENCES 1. Baker, G., J.H. Pepper, L.H. Johnson, and E. Hastings, J. Insect Physiol. 5:47 (1960). 2. Soliday, C.L., G.J. Blomquist, and L.L. Jackson, J. Lipid Res. 15:399 (1974). 3. Nelson, D.R., and D.R. Sukkestad, Biochemistry 9:4601 (1970). 4. Nelson, D.R., D.R. Sukkestad, and R.G. Zaylskie, J. Lipid Res. 13:413 (1972). 5. Borror, D.J., and D.M. DeLong, "An Introduction to the Study of Insects," Holt, Rinehart, and Winston, New York, NY, 1971, p. 122. 6. Tartivita, ICA., and L.L. Jackson, Lipids 5:35 (1970). 7. Jackson, L.L., Ibid. 5:38 (1970). 8. Jackson, L.L., Comp. Biochem. Physiol. 41B:331 (1972). 9. Nelson, D.R., and D.R. Sukkestad, J. Lipid Res. 16:12 (1975). 10. Hutchins, R.F.N., and M.M. Martin, Lipids 3:250 (1968). 11. Blomquist, G.J., T. Blailock, R.W. Scheetz, and L.L. Jackson, Comp. Biochem. Physiol. (In press). [ R e c e i v e d S e p t e m b e r 29, 1975 ]
In Vitro Activity of the Fatty Acyl Desaturases of Human Cancerous and Noncancerous Tissues ABSTRACT
T h e m i c r o s o m a l d e s a t u r a s e a c t i v i t y of human cancerous and noncancerous t i s s u e s was m e a s u r e d i n v i t r o using 1-14C,- 1 1 , 1 4 - e i c o s a d i e n o i c a n d 1-14C oleic acids as substrates. Tissues used were a case o f ovarian cancer, a u r i n a r y b l a d d e r cancer, a rectal cancer, a n d a n o n specific c o l o n i c ulcer w i t h a p p r o p r i a t e l y n o r m a l tissues. W h e n 1 1 , 1 4 - 2 0 : 2 was used as s u b s t r a t e , r a d i o a c t i v e t e t r a e n e a n d t r i e n e were p r o d u c e d . T h e t e t r a e n e was i d e n t i f i e d b y r a d i o gas c h r o m a t o g r a p h y as a r a c h i d o n i c acid ( 5 , 8 , 1 1 , 1 4 - 2 0 : 4 ) , a n d the triene had a retention time of 5 , 1 1 , 1 4 - 2 0 : 3 . T h u s , t h e possibility arises t h a t a A8 desaturase was involved. I n t h e A 6 desaturase, w i t h t h e u r i n a r y b l a d d e r
c a n c e r o u s tissue, t h e d e s a t u r a s e a c t i v i t y a p p e a r e d t o be d e c r e a s e d in c o m p a r i s o n t o n e i g h b o r i n g tissue, w h e r e a s w i t h t h e c o l o n i c c a n c e r tissue, t h e desaturase appeared t o b e relatively increased, t h o u g h t h e n u m b e r of samples was ina d e q u a t e for c o n f i d e n c e . INTRODUCTION
T h e a t t a c k o n c a n c e r c a n b e carried o u t o n l y w i t h t h o r o u g h k n o w l e d g e of its m e t a b o l i c characteristics. One of t h e p r o p e r t i e s o f r a p i d l y dividing ceils is t h e n e e d for u n s a t u r a t e d f a t t y acids as c o m p o n e n t s of t h e p h o s p h o l i p i d s n e c e s s a r y for t h e f o r m a t i o n o f cell m e m b r a n e s . T h e s o u r c e o f t h e s e f a t t y acids a n d t h e i r m e t a b o l i s m w i t h i n t h e m a l i g n a n t cell t h u s becomes a matter of considerable importance. LIPIDS, VOL. 11, NO. 1
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In particular, the various abilities of the malig- N NH4OH and 0.15 ml 1% Triton) in a nant cells to transform the precursor fatty total volume of 4 ml 0.15 M KC1 and 0 . 2 5 M a c i d s - oleic, linoleic, and linolenic - into the sucrose. The final pH of the solution was 7.1. 609, 6o6, and 603 polyunsaturated fatty acids The vials were shaken for 30 min at 37 C, and apparently required by membrane phospho- the reaction was stopped by addition of 0.1 ml lipids may represent a difference from normal of 9N H 2 SO4. The total lipid was extracted by cells. Chiappe et al. (1) have reported that both the method of Bligh and Dyer (4). After the A9 and A6 desaturases are depressed in two evaporation of the chloroform at 40 C under rat hepatomas as compared with normal liver N2, the lipids were subjected to methanolysis tissue. Because the A5 desaturase may be under with 2 ml of 5% methanolic HC1 (for up to d i f f e r e n t regulation and because different 50 mg) for 30 min at 85 C. The methyl esters tumors in different species may have distinct were extracted with pentane, washed with characteristics, the various desaturases were water, and freed from solvent. One portion of investigated in several human cancers. the methyl ester mixture was separated on t 0% AgNO3-impregnated silica gel plates (5) using 5% acetone in toluene as developing solution. MATERIALS AND METHODS Areas containing fatty acids with different 1 _1 4C_ 11,14-eicosadienoic acid was pur- degrees of unsaturation were located by spraychased from DHOM Products, Ltd. (North ing with an ethanolic solution of 2 , 7 -dichloroHollywood, CA) and 1-14C-oleic acid from fluorescein and by comparison with authentic New England Nuclear Corp. (Boston, MA). standards (Nu-Chek Prep, Inc., Elysian, MN). Both substances were over 99% pure by thin Each fraction was scraped off the plate, and the layer chromatography (TLC) and radio gas radioactivity in each area was measured in the liquid chromatography (GLC) and did not con- l i q u i d s c i n t i l l a t i o n spectrometer (Packard, tain any of the metabolic products concerned. Model 3003). Tissues studied in this research were a case A second portion of the methyl ester mixof chronic nonspecific ulcer (21 year old male), ture was separated by TLC as described above, a case of ovarian cancer (51 year old female), a and each fraction was further analyzed by radio case of urinary bladder cancer (61 year old gas chromatography using the Packard gas female), and a case of rectal cancer (75 year old chromatograph with gas proportional counter male). model 894, fitted with a 6 f t x 4 mm glass Tissues were collected during surgical pro- column packed with Apolar 10C (Applied cedures under sterile conditions at the City of Science Laboratory, State College, PA) and Hope Medical Center (Duarte, CA) and were operated at 185 C column temperature. Peaks placed immediately in ice cold Hank's solution. were identified, where possible, by comparison A portion of each specimen was subjected to with authentic standards. histological examination to confirm the malignant or normal condition of the tissues. All RESULTS procedures were carried out at 4 C unless stated otherwise. With 20:2606 as substrate, radioactivity was Each tissue was homogenized with 4 parts found in the areas of the TLC plate corre(v/w) of homogenizing buffer (2), and the sponding to diene, triene, and tetraene (Table homogenate was centrifuged at 600 x g for 30 I). The tetraenoic acid was identified as arachim i n . T h e supernatant was centrifuged at donic (5,8,11,14-20:4) by comparison of its 9,000 x g for 30 min, and the resulting super- GLC retention time with that of an authentic natant was centrifuged at 105,000 g for 90 min. standard. The retention time of the single comThe pellet from the last centrifugation, the ponent of the triene zone was the same as that microsomal fraction, was suspended in homog- of 5,11,14-20:3. Ozonolysis was carried out enizing buffer (2:1, v/v) with sonication, and with both fractions (6) but, because of the the protein content was measured by the small amounts involved, was not definitive. method of Lowry et al. (3). Including the buf- Throughout the experiment, the total radiofer solution, each vial contained 3-10 mg of activity of tetraene was higher than that of microsomaI protein and the following addi- triene. tions, in /amoles: adenosine triphosphate, 8; In Table II can be seen the results of expericoenzyme A, 0.4; reduced nicotinamide ade- ments designed to test the activity of the A6 nine dinucleotide, 5; KF, 6; nicotinamide, 2; desaturase. In the urinary bladder cancer, MgC12, 7.5; glutathione, 2.2: phosphate enzyme activity, as measured by diene producbuffer, pH 7.3, 62; and 1 pc (1 pm) of tion per mg microsomal protein, appeared to be substrate fatty acid (in solution in 0.05 ml 0.1 decreased as compared with that of normal t
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t
.
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11,14-20:2
20:3
Normal colonic
83
Ovarian cancer
92
3
Urinary bladder cancer
90
4
Urinary bladder normal
92
Colonic
83
5,8,11,14-20:4
7
10 1.9 b
2.7 b 5
0.7 b
1.1 b 6
0.2 b
0.4 b
1.1 b
1.8 b
3
6
11 1.8 b
cancer
Normal colonic
87
3.1 b
6
7 0.7 b
0.7 b
aEach vial contained, in 4 ml, 3-10 mg microsomal protein and the following additions, in /zmoles: adenosine triphosphate, 8; coenzyme A, 0.4; reduced nicotinamide adenine dinucleotide, 5; potassium fluoride, 6; nicotinamide, 2; MgC12, 7.5; glutathione, 2.2; phosphate buffer, pH 7.3, 62; and 1 /~c (1 /am) of substrate fatty acid (in solution in 0.05 ml 0.1 N NH4OH and 0.15 ml 1% triton). Vials were incubated 30 min at 37C, and fatty acid methyl esters were prepared and quantitated using radio gas chromatography. bExpressed as percent of radioactive fatty acids produced per mg of microsomal protein. s u r r o u n d i n g tissue. In t h e c o l o n i c c a n c e r , h o w ever, t h e A 6 d e s a t u r a s e a c t i v i t y a p p e a r e d to be increased.
TABLE 11 Products Formed from 1-14C-Oleic Acid on Incubation with Microsomal Fractions from Human Cancerous and Normal Tissuesa
DISCUSSION Following i n c u b a t i o n of 11,l 4-eicosadienoic acid w i t h t h e m i c r o s o m a l f r a c t i o n f r o m h u m a n c a n c e r o u s a n d n o n c a n c e r o u s t i s s u e s , radioa c t i v i t y was f o u n d in diene, t r i e n e , a n d t e t r a e n e f a t t y acids. T h e m e t h y l e i c o s a t e t r a e n o a t e h a d t h e s a m e G L C r e t e n t i o n t i m e as m e t h y l arachid o n a t e a n d was p r o b a b l y i d e n t i c a l w i t h t h a t acid. T h e m e t h y l ester of t h e t r i e n o i c f a t t y acid h a d a r e t e n t i o n t i m e c o r r e s p o n d i n g t o t h a t of 5,1 1,1 4-20: 3, t h e e x p e c t e d p r o d u c t f r o m a liver s y s t e m (7), b u t c o u l d n o t be f u r t h e r i d e n t i f i e d b e c a u s e o f t h e small a m o u n t of p r o d u c t available. T h e diene z o n e y i e l d e d o n l y t h e s t a r t i n g s u b s t r a t e , 20: 2606, a n d t h e 1-14C-1 1,1 4-eicosad i e n o i c acid u s e d in t h e e x p e r i m e n t was s h o w n t o be p u r e b y radio G L C a n d n o t to c o n t a i n a n y r a d i o a c t i v e 18: 2. T h u s , t h e r e is a n e e d t o e x p l a i n t h e origin o f t h e a r a c h i d o n a t e . In t h e i n c u b a t i o n s y s t e m , K F was u s e d t o i n h i b i t c h a i n e l o n g a t i o n , a n d r e t r o c o n v e r s i o n o f t h e 1 1 , 1 4 - 2 0 : 2 t o 9 , 1 2 - 1 8 : 2 followed by elongation and desaturation to a r a c h i d o n a t e (8) was s h o w n t o be u n l i k e l y b e c a u s e a trial e x p e r i m e n t u s i n g 1 - 1 4 C - 1 8 : 2 did n o t yield a r a c h i d o n a t e u n d e r t h e s e c o n d i t i o n s .
Radioactive Fatty Acids Isolated (%) Tissue
9-18:1"
6,9-18:2
Urinary bladder cancer
97.5
Urinary bladder normal
99
Colonic cancer
98.8
1.2
Normal colonic
98.8
1.2
2.5 0.14 b 1.0 0.38 b
0.34 b 0.14 b
aEach vial contained, in 4 ml, 3-10 mg microsomal protein and the following additions, in ~zmoles: adenosine triphosphate, 8; coenzyme A, 0.4; reduced nicotinamide adenine dinucleotide, 5; potassium fluoride, 6; nicotinamide, 2; MgC12, 7.5; glutathione, 2.2; phosphate buffer, pH 7.3, 62; and 1 /~c (1 #m) of substrate fatty acid (in solution in 0.05 ml 0.1 N NH4OH and 0.15 ml 1% triton). Vials were incubated 30 min at 37C, and fatty acid methyl esters were prepared and quantitated using radio gas chromatography. bExpressed as percent of radioactive f a t t y acids produced per mg of microsomal protein. T h e a l t e r n a t i v e p a t h w a y s are (a) c o n v e r s i o n o f 11,14-20:2 to 8,11,14-20:3, which has been s h o w n t o p r o c e e d readily t o a r a c h i d o n a t e , a n d LIPIDS, VOL. 11, NO. 1
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(b) c o n v e r s i o n of 1 1 , 1 4 - 2 0 : 2 t o 5 , 1 1 , 1 4 - 2 0 : 3 a n d c o n v e r s i o n of t h i s acid t o a r a c h i d o n a t e , as has b e e n suggested b y Takagi (9) f r o m in vivo e x p e r i m e n t s w i t h rats b u t d e n i e d b y S c h l e n k et
al.
(10).
Recently, S p r e c h e r a n d Lee (7) have carried o u t e x p e r i m e n t s with rat liver m i c r o s o m e s t h a t a p p e a r t o d e n y t h e e x i s t e n c e o f a A 8 desaturase a n d t h e i r results have b e e n c o n f i r m e d in this l a b o r a t o r y . In o u r e x p e r i m e n t s w i t h h u m a n tissues, h o w e v e r , m o r e t h a n 10% of s u b s t r a t e 1 1 , 1 4 - 2 0 : 2 was c o n v e r t e d t o a r a c h i d o n a t e in s o m e cases, a n d e i t h e r of t h e possible r o u t e s w o u l d r e q u i r e d e s a t u r a t i o n at t h e 8 p o s i t i o n . As N a k a z a w a et al. have r e p o r t e d previously ( 1 1 ) , the p h o s p h o l i p i d a n d triglyceride f a t t y a c i d c o m p o s i t i o n o f b i o p s i e d h u m a n liver tissues were distinctly d i f f e r e n t f r o m t h o s e o f c o r r e s p o n d i n g rat tissues, a n d it is possible to c o n s i d e r t h e e x i s t e n c e of a A8 desaturase in h u m a n tissue. F u r t h e r s t u d y will b e n e c e s s a r y t o clarify t h e r o u t e f r o m 20:2006 t o arachid o n a t e a n d t h e co6 p a t h w a y in h u m a n cancerous a n d n o r m a l tissues.
ICHIRO NAKAZAWA T h i r d D e p a r t m e n t of I n t e r n a l M e d i c i n e School o f Medicine Tohoku University Sendal, J a p a n J A M E S F. M E A D D e p a r t m e n t o f Biological C h e m i s t r y a n d L a b o r a t o r y of N u c l e a r M e d i c i n e a n d R a d i a t i o n Biology University o f California Los Angeles, California 9 0 0 2 4
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R O B E R T H. Y O N E M O T O D e p a r t m e n t of O n c o l o g i c a l Surgery City of H o p e Medical C e n t e r D u a r t e , California 9 1 0 1 0 ACKNOWLEDGMENTS These studies were supported in part by Contract E(O4-1)GEN-12 between ERDA (author, please spell out) and the University of California; by US Public H e a l t h Service R e s e a r c h C a r e e r Award No. GM-K-6-19,177 from the Division of General Medical Sciences, National Institutes of Health; by a grant from the Chancellor's Research Opportunity Fund, UCLA; and by funds from the UCLA General Support Grant. G.A. Dhopeshwarkar and C. Subramanian of this Laboratory provided advice and aid in this research. REFERENCES 1. Chiappe, L.E., M.E. De Tomas, and O. Mercuri, Lipids 9:489 (1974). 2. Brenner, R.R., and R.O. Peluffo, J. Biol. Chem. 241:5213 (1966). 3. Lowry, O.H., N.J. Rosebrough, A.L. Farr, and R.J. Randall, Ibid. 193:265 (1951). 4. Bligh, E.G., and W.J. Dyer, Can. J. Biochem. Physiol. 37:911 (1959). 5. Applied Science Gas Chrom. News Letter, Vol. 13, No. 1, Jan/Feb (1972). 6. Stein, R.A., and N. Nicolaides, J. Lipid Res. 3:476 (1962). 7. Sprecher, H., and C. Lee, Biochim. Biophys. Acta 388:113 (1975). 8. Stearns, E.M., Jr., J.A. Rysavy, and O.S. Privett, J. Nutr. 93:485 (1967). 9. Takagi, T., Bull. Chem. Soc. Jap. 38:2055 (1965). 10. Schlenk, H., D.M. Sand, and J.L. Gellerman, Lipids 5:575 (1970). 11. Nakazawa, I., and S. Yamagata, Tohoku J. Exp. Med., 103:129 (1971). [ R e c e i v e d S e p t e m b e r 15, 1 9 7 5 ]
