Incorporation of 1-14C-Acetate into C26 Fatty Acids of the Marine Sponge Microciona prolifera REGINALD W. MORALES and CARTER LITCHFIELD, Department of Biochemistry, R utgers University, New Brunswick, NJ 08903

ABSTRACT

A d e n o s i n e triphosphate (ATP), reduced nicotinamide adenine dinucleotide (NADH), r e d u c e d nicotinamide adenine dinucleotide p h o s p h a t e (NADPH), coenzyme A (CoA), glutathione, and streptomycin sulfate were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium 1-14C-acetate and 1-14C-oleic a c i d were obtained from Applied Science Laboratories (State College, PA).

The incorporation of 1A4C-acetate into the many fatty acids of the marine sponge Microciona prolifera was investigated. Probable precursors of 26:2A5,9 and 26:3A5,9,19 showed high levels of radioactivity, supporting the following pathways for the biosynthesis of C96 acids: 16:0--, ~

Incubation Conditions

26:0--, 26:1A9~ 26:2A5,9

Incubation conditions for sponge experiments were selected to resemble the environment at the collection site as much as possible. F o r the intact sponge systems, sponge sampies were carefully cleaned and the tips o f the fingedets, 0.2-1.5 g blotted wet weight, were placed in flasks containing 25 ml seawater and 10-25 /aCi 1-14C-acetate (55 /aCi/mole). The seawater was collected at the sampling site, filtered through 0.22 /am Millipore filters, and buffered at its original pH (c. 8.1) with 0.02 M

16:1A9 ~-'* ~ 26:1A19 ~ 26:2A9,19--" 26:3A5,9,19 Degradation of the unsaturated C26 acids at their double bonds showed that the ] 4 C was concentrated near the carboxyl end of the chain. Hence, chain elongation was the major mechanism for acetate incorporation into these acids. INTRODUCTION

Our recent investigations ( 1 , 2 ) o f fatty acids in the marine sponge Microciona prolifera revealed that 48% of its acids have C24-C28 Qhain lengths. Among these are an unusual new family o f polyunsaturated fatty acids cont a i n i n g n o n m e t h y l e n e interrupted double bonds. Specific structures identified included: (major components) 26:2A5,9; 26:3A5,9,19 and (minor components) 24:2A5,9; 25:2A5,9; 26:3A5,9,17; 27:3A5,9,19; 27:3A5,9,20. Bios y n t h e t i c p a t h w a y s for such acids were proposed (2) based on probable intermediates found in our fatty acid analyses. We have now completed a series of 14C~cetate incorporation studies on Microciona fatty acids. These experimental results provide direct evidence on how 26:2A5,9 and 26:3A5,9,19 are synthesized by this sponge. EXPERIMENTAL PROCEDURES Materials

Microciona prolifera sponge colonies were collected off Barley Point, Navesink, NJ at a depth of % 1 m during the summer and fall of 1975. The sponges were transported to the laboratory in seawater and used within 2 hr after collection.

tris-(hydroxymethyl)-aminomethane-HC1. F o r the whole cell systems, sponge tips were placed in Ca ++ and Mg§ free seawater, and the cells were disaggregated according to Humphreys (3). The cells were then washed, centrifuged, and the pellet resuspended in a seawater medium identical to that used for the intact sponge incubations. F o r the cell-free systems, sponge cell pellets were resuspended in 5 volumes o f 0.55 M sucrose containing 20 mM phosphate buffer (pH 7.4) and homogenized using a mechanically d r i v e n T e f l o n - o n - g l a s s homogenizer. Cell rupture was monitored with a light microscope. Unbroken cells and debris were removed by centrifugation at 600 x g. The incubation mixture for acetate incorporation contained 6 /amoles MgC12, 40/amoles NaHCO3, 20/amoles ATP, 2/amoles CoA, 4/amoles NADH, 4/amoles NADPH, 2/amoles glutathione, 40/amoles phosphate, 1.1 mmoles sucrose, 25 /aCi 1-14C acetate, and 11 mg sponge protein [assayed by microkjeldahl analysis (4)] in a total volume of 2.0 ml. All incubations were carded out in Dubnoff metabolic shakers at temperatures similar to those o f the seawater at the collection site ( 1 6 - 2 6 C, d e p e n d i n g on time of year). Streptomycin was added (50/ag/ml) to prevent

570

C26 FATTY ACIDS OF A MARINE SPONGE bacterial growth. Incubations were stopped at the desired times by homogenization in 20 volumes of 2:1 chloroform-methanol. Fatty Acid Analysis

Total lipid was extracted according to the method of Folch et al. (5). F a t t y acid methyl e s t e r s were prepared by H2SO4-catalyzed methanolysis (6) and isolated by thin layer chromatography (TLC). When nonradioactive carriers were necessary, fatty acids isolated previously from Microciona (2) were used. Total methyl esters were fractionated according to degree of unsaturation by TLC on AgNO3-impregnated silicic acid (Ag+-TLC)and then according to chain length using preparative gas liquid chromatography (GLC) as described previously (2). This procedure separates Microciona fatty acids not only according to chain length and number of double bonds but also according to the position of those double bonds in certain fatty acid chains. A detailed discussion of this effect has been given elsewhere (2); however, the 26:1 isomers can be cited here as a typical example. All the 26:1A9 present appeared in the monoenes II band, while the 26:1 A17 and 26:1 A19 isomers were recovered from the monoenes I band. Hence, the individual chain lengths isolated from each band represented either single or very similar (r + 609) positional isomers. Aliquots taken at various stages of the fractionation were evaporated and dissolved in a toluene scintillator (Omniscint, ICN Pharmaceuticals, Inc., Cleveland, OH) for radioassay. All samples were counted in a Beckman LS-230 liquid scintillation counter. Samples with low activity were counted for sufficient time to produce 20 standard deviation of <5%. Distribution of 14C Along C26 Chains

Unsaturated C26 methyl esters resolved by preparative TLC + GLC were converted to dinitrophenylhydrazones by a procedure adapted from Privett and Nickell (7). About 0.5 ml CH2C1 z saturated with ozone at - 7 0 C was added to 0.5 ml CH2C12 containing 30 to 100 /ag of individual radioactive C26 methyl esters. Excess ozone was immediately removed b y bubbling He gas through the solution. Then 2 mg of triphenylphosphine followed by 1 mg of dinitrophenylhydrazine (DNPH) were added. The reaction mixture was stirred in a vortex mixer, its volume reduced under a stream of He, and then quantitatively transferred to 5 x 20 cm silica gel precoated plates (Quantum Industries, Fairfield, NJ) for TLC. Best results were obtained when the elapsed time between ozonolysis and start of TLC was kept under 5

571

rain. Reaction conditions were optimized using methyl 1-14C-oleate until yields />70% were attained. The alkyl-hydrazones, alkyl-dihydrazones, and alkyl-ester,hydrazones produced from the reactions of C26 unsaturates were then isolated by TLC using solvent systems ranging from 75:25 to 100:0 benzene-diethyl ether. Slight changes in the diethyl ether content of the developing solvent produced large changes in Rf values of the individual hydrazones, permitting clean separation of the desired hydrazones and elimination of the reaction by-products. Bands were recovered and rechromatographed at least three times until a constant specific activity was obtained. Bands were identified by cochromatography with hydrazones of known structure, which were produced by reactions with methyl oleate, methyl 6-octadecenoate, various 0~-olefinic h y d r o c a r b o n s , 1,5-hexadiene, 1,7-octadiene, 1,9-decadiene, 1,11-dodecadiene, and methyl esters from Limnanthes douglasff (8). Relative amounts of the hydrazone compounds recovered from TLC were determined by measurement of absorbance at 358 nm (9) in CHCI3. The samples were then radioassayed in the same manner described earlier, except that the minor quenching o f 14 C measurement by the small amount of hydrazone used was taken into account for all calculations. Division of the measured radioactivity in each fragment by the corresponding absorbance for each hydrazone yielded the relative specific activities from the different parts o f a given fatty acid chain. Calculations included corrections for the n u m b e r of hydrazones per fragment and normalization of values within each chain. One drawback o f this h y d m z o n e derivatization technique was the precipitation of short chain dihydrazones (~
Optimization studies of 14C.acetate incorporation into the ultra long chain fatty acids of Microciona were carried out using both in vivo LIPIDS, VOL. 12, NO. 7

572

REGINALD W. MORALES AND CARTER LITCHFIELD TABLE I Distribution o f 14C Radioactivity Among C22, C24, and C26 Fatty Acids Containing 0-3 Double Bonds Percentage of fatty acid radioactivityb

C22

C24

C26

Intact sponge

14.2

Whole cells

17.8 17.6

22.2 27.3 29.8

24.2 15.1 6.0

System a

Cell-free homogenate

aEach system was incubated for 3 hr at 26 C with 25 ,ttCi 1-14C-acetate. A single sponge sample was used to provide aliquots for all three systems. Other conditions and preparation o f systems are given in text. bTotal fatty acid radioactivity = 100%.

i--

200

oo,,, o I00

0.0

0.5

1.0

1.5

m MOLES ACETATEPER LITER OF INCUBATIONMIXTURE

Since nothing was known about the uptake of soluble nutrients such as acetate by Microciona, the effect of acetate concentration upon acetate uptake was investigated (Fig. 1). Above 0.2 mM concentration, the rates of acetate incorporation into both the total fatty acids and into the C26 acids appeared to be fairly constant, although the relative amount of radioactivity in the C26 acids dropped slightly at higher acetate concentrations. Therefore, all subsequent investigations were run with the acetate concentration between 0.2 and 0.3 raM.

FIG. 1. Effect of acetate concentration in the Distribution of 14C Among Fatty Acids incubation medium on the incorporation of 14rC. To determine the distribution of radioacetate into Microciona prolifera fatty acids. Intact activity among the major fatty acids after sponge incubated 10 hr at 20C with 10-20 ~tCi acetate incorporation, 5.3 g of intact sponge 1J 4C-acetate. was incubated for 4 hr at 16 C with 25/aCi of 14C-acetate. This larger sample size allowed and in vitro systems. Since almost half of this subsequent separations to proceed without the organism's fatty acids are C24-C28 , the distri- addition of nonradioactive carriers. Fatty acid bution of radioactivity according to chain methyl esters prepared from this sample were length, specifically among the >.~C22 acids, was then separated using Ag+-TLC followed by our initial concern. preparative GLC. Gas chromatograms of the Table I shows the distribution of 14C in the individual TLC bands confirmed band identifi(222, C24 , and C26 acids for three different cations and showed mass distributions of fatty incubation systems acting on aliquots of the acids quite similar to those reported earlier (2). same sponge sample. All three systems had the Radioactivity measurements for the various capacity to incorporate relatively high amounts fractions separated are reported in Table II. o f acetate into the longer chain lengths. Results for all C26 acids were further conAlthough the distribution of radioactivity for firmed by a second analysis in which the 1222 and C24 acids was relatively equal in all sequence of Ag+-TLC and GLC separations was three systems, there was a signifiant drop in the reversed. C26 radioactivity in the whole-cell and cell-free An unexpected finding in this current fracsystems. This lower incorporation of ] 4C into tionation of Microciona fatty acids was that the the C26 acids was independent of incubation C26 material from the dienes II band could be time and various manipulations of reaction con- further separated by Ag+-TLC into two bands. d i t i o n s ; alterations in the homogenization The very minor ( * 0 . 7 wt %), faster moving, protocol, use of different buffers, and changes second band was isolated and identified as in types and levels of cofactors did not increase 26:2A9,19 based on GLC analysis of the it. Since maximum 14 C incorporation into the aldehydic products from ozonolysis and TLC C26 acids was most desirable for our C26 bio- i d e n t i f i c a t i o n of their DNPH hydrazone synthetic studies, all further experiments were derivatives. This 26:2A9,19 isomer had not run using the intact sponge system. been found previously (2). A possible explanaLIPIDS, VOL. 12, NO. 7

C26 FATTY ACIDS OF A MARINE SPONGE

573

TABLE II Distribution of 14C Radioactivity Among Mierociona prolifera Fatty Acids a % of Fatty acid radioactivity b Chain length

Saturates

Monoenes I II

Dienes I

II

Trienes

Total

16

3.1

0.1

2.8

tr

tr

tr

6.0

18 20 22 24 26 Others c

4.7 3.2 4.3 2.9 0.5 6.6

0.1 0.7 4.8 14.7 11.1 1.8

12.9 1.7 0.3 0.2 6.0 1.3

tr tr 0.1 0.8 1.6 0.6

0.1 0.3 0.5 0.6 4.2 d 0.6

tr 0.4 0.8 1.4 1.8 e 1.0

17.8 6.3 10.8 20.6 25.2 11.9

alntact sponge incubated 4 hr at 16 C with 25/~Ci of 1-14C-acetate. bTotai fatty acid radioactivity = 100*7~ Tetraenes + pentaenes + hexaenes = 1.4%. cC14,C28, branched chain, and odd carbon number fatty acids. dFurther separated by Ag+-TLC into 3.3% 26:2A9,19 and 0.9% 26:2~5,9. eFurther separated by Ag+-TLC into 1.6% 26:3A5,9,19 and 0.2% 26:3r t i o n f o r its a p p e a r a n c e h e r e m i g h t b e t h e seasonal c h a n g e s in 2 6 : 3 A 5 , 9 , 1 9 c o n t e n t o f M i c r o c i o n a tissues ( I 1) a n d t h e p r o b a b l e role o f 2 6 : 2 A 9 , 1 9 as a p r e c u r s o r o f 2 6 : 3 A 5 , 9 , 1 9 (2). T h e s a m p l e a n a l y z e d earlier ( 2 ) was c o l l e c t e d in early s u m m e r w h e n 2 6 : 3 A 5 , 9 , 1 9 levels are lower, whereas t h e s a m p l e a n a l y z e d in this study was harvested in late fall w h e n 2 6 : 3 A 5 , 9 , 1 9 levels are higher. T h e overall d i s t r i b u t i o n o f 14 C r a d i o a c t i v i t y a m o n g M i c r o c i o n a f a t t y acids a c c o r d i n g t o chain length and unsaturation indicates that the e n z y m e s y s t e m for c h a i n e l o n g a t i o n o f f a t t y acids is especially active in this organism. Over 80% o f t h e t o t a l 14C was i n c o r p o r a t e d i n t o C18-C26 acyl chains t h a t are n o r m a l l y considered t o be p r o d u c t s o f c h a i n e l o n g a t i o n ; a n d over 4 5 % o f t h e t o t a l 14C was f o u n d in t h e C24 a n d C 26 chains. We r e c e n t l y p r o p o s e d (2) b i o s y n t h e t i c p a t h ways f o r 2 6 : 2 A 5 , 9 a n d 2 6 : 3 A 5 , 9 , 1 9 i n Microciona b a s e d u p o n k n o w n p a t t e r n s o f f a t t y acid biosynthesis (12-15) and upon the presence of p r o b a b l e p r e c u r s o r acids i n this organism. T o e v a l u a t e t h e s e proposals, t h e t o t a l 14 C f o u n d i n e a c h o f t h e p r e c u r s o r a n d p r o d u c t acids a n d t h e i r relative specific activities (% o f t o t a l f a t t y acid 14C/% o f t o t a l f a t t y acid mass) have b e e n t a b u l a t e d in Tables III a n d IV. A l m o s t all o f t h e postulated precursors and their unsaturated C26 p r o d u c t s c o n t a i n e d s u b s t a n t i a l levels o f t o t a l r a d i o a c t i v i t y . A l t o g e t h e r , t h e f a t t y acids listed i n Tables III a n d I V a c c o u n t e d f o r over 79% o f t h e t o t a l f a t t y acid 14C f o u n d , indicating t h a t t h e y are u n d e r g o i n g active biosynthesis. B o t h t h e s e f i n d i n g s s u p p o r t t h e proposed pathways. T h e specific activities o f t h e acids in T a b l e s III a n d I V s h o w t h a t t h e p r e c u r s o r acids ( 1 6 : 0

TABLE IIl Radioactivity Found in Fatty Acids of Proposed Pathway for 26:2A5,9 Biosynthesis in Microeiona proliferaa Fatty acid 16:0 18:0 20:0 22:0 24:0 26:0 26:1A9 26:2A5,9

% of Total fatty acid 14C 3.1 4.7 3.2 4.3 2.9 0.5 6.0 0.9

Specific activity 0.8 2.2 4.0 1.4 1.5 5.0 !0.0 0.05

alntact sponge incubated for 4 hr at 16 C with 25 ~tCi of 1-14C_acetat e. TABLE IV Radioactivity Found in Fatty Acids of Proposed Pathway for 26:3A5,9,19 Biosynthesis in Microeiona prolifera a Fatty acid 16:1A9 18:IAll 20:1A13 22:1A15 24:1A17 26:1A19 26:2A9,19 26:3A5,9,19

% of Total fatty acid 14C

Specific activity

2.8 12.9 2.4 4.8 14.7 11.1 3.3 1.6

4.0 3.4 4.0 9.6 3.9 2.5 4.7 0.1

alntact sponge incubated for 4 hr at 16 C with 25 #Ci of 1-14C.acetate. 2 6 : 1 A 9 a n d 1 6 : 1 A 9 ~ 2 6 : 2 A 9 , 1 9 ) were all labeled t o a similar degree. However, t h e specific activities o f p r o d u c t acids 2 6 : 2 A 5 , 9 a n d 2 6 : 3 A 5 , 9 , 1 9 were a n o r d e r o f m a g n i t u d e LIPIDS, VOL. 12, NO. 7

REGINALD W. MORALES AND CARTER LITCHFIELD

574 C26

ACID

CARBOXYLEND

METHYL END

,..J

z LIJ =E E: Ia.I Q..

x ILl

/

ELONGATION ONLY >n..

"IP-

D E H O V O "1" ELONGATION

I

99

II

TR

II

TR

I

I I I I I I . . I . I I HOOC*-C-C-C-C=C-C*-C-C " - - C - C - C - C - C - C - C - C - C - C = C - C - C - C - C - C - C H 3 60 I 40 I 0 I 0 I I I I I I I I I H 0 0 c3* C . ~ ~* C _ C * C_~*C_e*__C.e'_C.P'_C_e,C_P._C_~,. ~ - ~ ,, ,,,,- ,,- ,, _ C . , ,~,* C _ C * C _ , ,~,.CH I, 23 II 15 39 ,I 23

FIG. 2. Distribution of ~+C radioactivity along the chains of the unsaturated C26 fatty acids of Microciona prolifera. Numbers indicate percent of activity found in each fragment after cleavage of the double bonds. Total +C activity in each chain = 100%. Values for 26:2A5,9 represent limits of specific activity based on calculations described in text. less. This is not merely the result of dilution by the large pools of 26:2A5,9 and 26:3A5,9,19 present, since the total 14C found in these two acids was considerably lower than found in most of the precursor acids. This suggests that A5 desaturation, the last step in b o t h proposed pathways, occurs more slowly than the previous elongation and A9 desaturation steps. It should be noted that the monoene fractions assayed for the 26:3A5,9,19 pathway actually represented a mixture of the 6o7 and 6o9 isomers with 6o7>>6o9. Hence, the radioactivity data in Table IV represents the sum of b o t h 2 6 : 3 A 5 , 9 , 1 9 and 26:3A5,9,17 biosynthetic activity. We have previously pointed out the probable presence of such a parallel pathway for 6o9 acids in Microciona (2). The 14C distribution reported in Table II also provides information on the biosynthesis of two other groups of fatty acids in Microciona. T h e combined tetra-, penta-, and hexaenoic polyunsaturates had a low specific activity (0.06), implying that 4-6 double bond acids originated in the diet and are incorporated directly into the sponge lipids without further chain elongation. On the other hand, the C24 LIPIDS, VOL. 12, NO. 7

and C 26 dienes and trienes produced by elongation of linoleic and linolenic acids were apparently undergoing more active biosynthesis, since the 24:26o6, 26:2606, and 26:36o3 fractions showed specific activities of 2.7, 1.0, and 0.5, respectively. Distribution of 14C Along C 2 6 Chains

To determine the extent to which chain elongation and de n e r o biosynthesis pathways contribute to C26 fatty acid biosynthesis in Microciona, each unsaturated C26 acid was cleaved at its double bonds; and the relative specific activities of the resulting fragments were measured (Fig. 2). Since the ozone-triphenylphosphene-dinitrophenylhydrazine treatment used did not permit direct isolation of the C4 fragment between A5 and A9 in 26:2A5,9 and 26:3A5,9,19 (see above), its activity was determined by difference. F o r 26:3A5,9,19 this involved partial hydrogenation with hydrazine (26:3A5,9,19 ~ 26:1A5 + 26:1A9 + etc.) prior to ozonolysis, so that the relative specific activities of b o t h the C5 and C 9 alkyl-esterhydrazones could be measured and their difference (the C 4 fragment) calculated.

C26 FATTY ACIDS OF A MARINE SPONGE

With 26:2A5,9 there was an additional probl e m with trace amounts of high activity 26:2A9,19 in the low activity 26:2A5,9 sample, probably due to tailing of the faster-moving 26:2A9,19 band when the two compounds were separated by Ag+ TLC. Hence, determinations of radioactivity in the C 9 ester-hydrazone from partially reduced 26:2A5,9 produced inaccurate results. As an alternative, the radioactivity of the A5-A9 segment was estimated from the C 17 and C 21 alkyl-hydrazones derived from the methyl end of partially reduced 26:2A5,9. These C 17 and C 21 hydrazones were partially separated by TLC, and the relative specific activities of the two fractions were measured. Using the equation C4 = C21 - C17, m i n i m u m and maximum C4 specific activities were calculated for both TLC fractions ass u m i n g a b r o a d compositional range of 0%~90% and 10%<~C21/>100% (mole %). Combining the higher m i n i m u m with the lower maximum gives the narrow range of relative specific activity for the C 4 fragment reported in Figure 2. Similar m a x i m u m / m i n i m u m calculat i o n s o n u n f r a c t i o n a t e d C21/C17 alkylhydrazone mixtures from two different hydrazinc hydrogenations of 26:2A5,9 (one to high 26:1 content, the other to low 26:1 content) yielded almost identical results for the specific activity range of the C 4 fragment. The distribution of 14C in the 26:1A9 and 26:2A5,9 chains was quite similar. 95-99% of the radioactivity was located in the first nine carbon atoms at the carboxyl end of the chain, indicating that the chain elongation pathway was the main route for 14C_acetate incorporation into these acids. However, the 14C in the 26:2A5,9 was more concentrated in the first five carbons at the carboxyl end than one would expect from a simple 16:0 -~ 26:0 elongation process. Apparently elongation from precursors longer than C16 occurred, indicating that the chain elongation system of Microciona will accept preformed C20-C22 substrate acids. The above findings are in agreement with our proposed 16:0 --> 26:2A5,9 biosynthetic pathway, and they strongly support a precursor/ product relationship for 26:1A9 and 26:2A5,9. T h e d i s t r i b u t i o n o f r a d i o a c t i v i t y in 26:1A19, 26:2A9,19, and 26:3A5,9,19 also pointed to chain elongation as the main process f o r 1-14C-acetate incorporation into these acids. 96% of the 14C in 26:1A9 was found in the 19 carbon segment at the carboxyl end of the chain. The 26:2A9,19 and 26:3A5,9,19 contained 99% and 82%, respectively, of their 14 C activity in the first nine carbons of the chain. As with 26:2A5,9, the 14C in the first five carbons of 26:3A5,9,19 was considerably

575

greater than in the A5-A9 fragment (58% vs. 24%), indicating chain elongation of ~C20 substrates. However, the presence of 18% radioactivity in the A9-A19 segment of the 26:3A5,9,19 chain was unexpected and puzzling. This could not be explained by full de novo biosynthesis of a C16 precursor, since the seven carbon unit at the methyl end of the chain contained no radioactivity at all. Chain elongation of a C14 or shorter precursor (14:0 --> 16:0 --> 16:1A9 -* --> 26:1A19 4--> 26:3A5,9,19) is a distinct possibility. Since none of the other C26 unsaturates were found to be similarly labeled, perhaps multiple systems for fatty acid elongation, such as those found in mouse brain (16), may occur in Microciona. From an overall point of view, however, the data of Figure 2 tend to support our proposed pathway for the biosynthesis of 26:3A5,9,19 in Microciona. The 14C labeling patterns in the 26:1A19, the 2 6 : 2 A 9 , 1 9 , a n d i n most (82%) of the 26:3A5,9,19 are as expected. Nevertheless, the presence of a minor alternative route for 26:3A5,9,19 production cannot be ruled out.

ACKNOWLEDGMENTS This investigation was supported in part by grants from the Rutgers University Research Council and the National Institutes of Health (PHS RR-7058). We thank Drs. T. van Es and Raju K. Pullarkat for their generous assistance and advice. REFERENCES 1. Jefferts, E., R.W. Morales, and C. Litchfield, Lipids 9:244 (1974). 2. M o r a l e s ,

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Biophys. Acta 431:206 (1976). 3. Humphreys, T., Develop. Biol. 8:27 (1963). 4. Niederl, J., and V. Niederl, "Micromethods of

5. 6. 7. 8. 9. 10. 11.

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Quantitative Organic Analysis," Wiley, New York, NY, 1942, pp. 69-78. Folch, J., M. Lees, and G.H.S. Stanley, J. Biol. Chem. 226:497 (1957). Johnston, P.V., "Basic Lipid Methodology," University of Illinois Press, Urbana, IL, 1971, p. 76. Privett, O.S., and E.C. Nickell, JAOCS 39:414 (1962). Smith Jr., C.R., M.O. Bagby, T.K. Miwa, R.L. Lohmar, and I.A. Wolff, J. Org. Chem. 25:1770 (1960). Haverkamp-Begemann, P., and K. de Jong, Rec. Tray. Chim. 78:275 (1959). Privett, O.S., and E.C. Nickeli, Lipids 1:98 (1966). Litchfield, C., and R.W. Morales, in "Aspects of Sponge Biology," Edited by F.W. Harrison and R.R. Cowden, Academic Press, New York, NY, 1976, pp. 183-200. Volpe, J.J., and P.R. Vagelos, Annu. Rev. Biochem. 42:21 (1973). Stumpf, P.K., Ibid. 38:159 (1969). LIPIDS, VOL. 12, NO. 7

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REGINALD W. MORALES AND CARTER LITCHFIELD

14. Brett, D., D. Howling, L.J. Morris, and A.T. James, Arch. Biochem. Biophys. 143:535 (1971). 15. Brenner, R.IL, Lipids 6:567 (1971). 16. Goldberg, L, I. Shechter, and K. Bloeh, Science

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182:497 (1973).

[Received December 6, 1976]