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Biosynthesis of docosa-4,7,10,13,16-pentaenoic acid in Euglena gracilis
Comp. Biochem. Physiol., 1977, Vol. 57B, pp. 261 to 263. Pergamon Press. Printed in Great Britain
BIOSYNTHESIS OF DOCOSA-4,7,10,13,16P E N T A E N O I C ACID IN E U G L E N A GRACILIS CLARENCE E, FOUCHE, JR.* AND JOHN G. CONIGLIO Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, U.S.A.
(Received 21 September 1976) Abstract--1. Polyenoic acids, including arachidonic and docosa-4,7,10,13,16-pentaenoic (22: 5n-6) acids, comprise a large portion of the total fatty acids of Euglena gracilis grown in the dark. 2. 14C from 1-[14C]-acetate, 1-[14C]-linoleate, and 1-[~4C]-arachidonate was incorporated into 22:5n-6 in cultures grown in the dark in the presence of these suhstrates. 3. 1-[x4C]-linoleate and 1-[14C]-arachidonate were elongated and desaturated to 5-[14C]-22:5n-6 and 3-[~4C]-22:5n-6, respectively, with little reutilization of acetyl units produced by oxidation. 4. l-[14C]-acetate was utilized to form the entire chain of 22:5n-6, with the carbons nearer the methyl group having a richer isotope content than the carbons nearer the carboxyl group. 5. Some 14C from 1-[14C]-acetate apparently entered the even carbon positions of the polyene by some unexplained process. INTRODUCTION
the Results section). The labeled compound was introduced into 2.8 1. Fernbach culture flasks containing 1 1. of Euolena broth. The medium was then sterilized by autoclaving, and 1 ml of EucJlena culture was added aseptically. The cultures were kept in the dark by wrapping the flasks with aluminum foil and grown for 2 weeks at room temperature. They were harvested by centrifugation at 5000 rev/min for 10min in 500-ml polycarbonate bottles, resuspended in distilled water and re-centrifuged. The pellet was dispersed in a small quantity of distilled water and transferred to a 50-ml flask, in which it was hydrolyzed in 40~o ethanolic KOH (using hyroquinone as anti-oxidant) for 2 hr under a stream of nitrogen. After extraction of non-saponifiable material, the hydrolysate was acidified and the free fatty acids recovered by extraction with petroleum ether. Methyl esters were prepared using the method of Metcalfe & Schmitz (1961). 14C determinations were made by liquid scintillation spectrometry in BraSs solution or in a toluene solution containing diphenyloxazole. Quenching was checked and corrections made by the use of an internal 14C toluene standard. Analytical gas-liquid chromatography was done with a column of SP-2340 (10~o on 100/120 mesh Supelcoport, Supelco, Inc., Bellefonte, PA). Gas-liquid radiochromatography was done with a similar column, the effluent of which passed through a heated tube to a continuous flow, proportional detector. For preparative collection of the labeled polyene, a column provided with a 1:1 mixture of 10~o SP-2340 on 100/120 mesh Supelcoport and 15~o EGSS-X on 100/120 mesh Gas Chrom P (Applied Science Laboratories, State Park, PA) was used, and the effluent was trapped on silanized glass wool. The collected methyl esters were eluted from the glass wool with petroleum ether. Analytical ga~liquid chromatography and gas-liquid radiochromatography were used to check the chemical and radiopurity of the collected samples. These were > 95~o pure for all samples used for characterization or degradation. Location of the 14C in fatty acid molecule was done on the hydrogenated derivatives (Farquhar et al., 1959) by the procedure of Dauben et al. (1953).
Polyunsaturated fatty acid metabolism in rat testis has been a major area of investigation in this laboratory for many years. Of particular interest has been the finding that the concentration of one particular polyene, docosa-4,7,10,13,16-pentaenoic acid (22: 5n-6), increases dramatically at the time of sexual maturation (Davies et al., 1966). Past investigations have included the biosynthesis of this compound (Davis & Coniglio, 1966) as well as some brief studies of its metabolism (Bridges & Coniglio, 1970). The latter type of study is difficult due to the non-availability of the pure compound, either radioactive or nonradioactive. The chemical synthesis is rather formidable (Kunau, 1971). The presence of relatively large amounts of this polyene in Euglena gracilis (Korn, 1964; Hulanicka et al., 1964) led us to investigate its biosynthesis from labeled substrates. Our studies show that the lipids of the organism contain from 7 to 10~o of its total fatty acids as 22:5n-6 when it is grown in the dark. Furthermore, it incorporates 14C from 14C-acetate, 14C_linoleat e and 14C_arachidonate into 22: 5n-6 in significant quantities. Chemical degradation studies of the biosynthesized polyene were done to determine the manner of labeling.
MATERIALS AND METHODS l-[14C]-sodium acetate and 1-[~4C]-linoleic acid were obtained from New England Nuclear Corporation, Boston, MA and l-[14C]-arachidonic acid from Dhom Products, Inc., North Hollywood, CA. Euglena gracilis, strain Z, was obtained from the Carolina Biological Supply House, Burlington, NC. Stock cultures of the organism were maintained under continuous flourescent light in screw-cap culture tubes containing Difco Euolena broth. Separate cultures of Euglena were grown containing the ~4C-substrates (amount of radioactivity used is given in
RESULTS About 7~o of the total fatty acids of Euglena grown in the dark was 22:5n-6. Arachidonic acid was pres-
* Present Address: Veterans Administration Hospital, Nashville, Tennessee 37232, U.S.A. 261
262
Q~I.ARENCE E. I~'Ot;CHI: JR. AND JOHN G. ('ONIGI.RJ
Table 1. Incorporation of labeled substrates into total fatty acids of Eu~/le~, Total fatty acids ",, of substratc 1 4 ( '
Fatty acid Sodium 1-[14C]-acetate Sodium 1-[14C]-linoleate ~ Sodium l-[14C]-arachidonate 'l
2(1" 8 47
"
__.~-_~,n-( of substratc laC ,v, 32
:' average of 4 cultures. " average of 3 cultures. results available on only 1 culture. d average of 3 cultures.
ent at a level of about 10')o of the total fatty acids, 22:6n-3 at about 3~;; and 20:5n-3 at about 5'~0. Other polyenes of 18-, 20-, and 22-carbon chain length amounted to 15°~o of the total fatty acids. In Table 1 is shown the amount of incorporation of ~4C from various substrates into total fatty acids of Ewlena and the amount of incorporation specifically into 22:5n-6. The most efficient incorporation was obtained with l"*C-arachidonate (32'!;, of the added substratee was incorporated into the isolated 22:5n-6). The highest specific activity obtained with each of these substrates was 992 ~Ci/mmole from 1-[~'*C]-acetate of specific activity 2.0mCi/mmole: 3.8 #Ci/mmole from 1-[~4C]-linoleate of specific activity 56mCi/mmole and 221 iLCi/mmole from 1-[~4C]-arachidonate of specific acitivity 5 8 m C i / mmole. The ~4C-activity not found in 22:5n-6 in the cultures incubated with l'~C-acetate was distributed in a number of fatty acids, both saturated and unsaturated. However, in the cultures incubated with
~4C-linoleate. no ~4C was observed in fatty acids of carbon chain length less than 18, while in those cultures incubated with 14C-arachidonate, the 14C was almost entirely in 22:5n-6 (aside from that remaining in 20:4n-6). The distribution of the labeled carbon in the 22:5n-6 isolated from cultures incubated with l-[~4C]-arachidonate or with l-[14C]-linoleate is shown in Table 2. In the former very little ~4C was present in the carboxyl {No. 1) or 2 (No. 2) carbon, while the third carbon had essentially all the activity of the molecule. In the case of the l-[~C]-linoleate substrate the fifth carbon had most of the ~4C activity while very little was present in any of the other carbons. The use of l-[14C]-acetate as substrate gave a more complex picture. The results of these incubations are shown in Table 3. Generally, the odd numbered carbons had most of the ~~C (compared to the even-numbered carbons). It is also apparent that the amount of 14C increased toward the methyl end of the molecule contrasted with the carboxyl end.
Table 2. Dauben degradation of 22:5n-6 synthesized in the presence of sodium l-[~aC]-arachidonatc or 1-[l~C]-linoleate
Carbon 11o.
Substrate 1-[l~C]-arachidonate
1-[~4C]-linoleate
Specific activity of benzoic acid {counts/min mmolc ~1 [B]
Initial specific activity (counts/min mmole ~C) EA]
I 2 3 1 2 3 4 5
9562 4667 1900 2871 1874 956 38(1 140
930 1623 3(I.666 471 113 378 288 1748
Table 3. Dauben degradation of 22:5n-6 synthesized from 1-[~a(']-acetate Ratio: Carbon no. l 2 3 4 5 6 7
Specific activity carboxyl carbon Specific activity average carbon in chain
Exp. 1
Exp. 2
Exp. 3
Exp. 4
0.91 0.13 1.72
1.18 0.05 0.40 0.89
0.88 0.04 1.30 0.29 1.35
0.77 0.42 0.76 1.37 0.94 1.79 2.98
Ratio [B]/[A] 0.1 0.4 16.1 (1.2 0.1 (1.4 (1.8 12.5
Docosapentaenoic acid synthesis in Euglena oracilis DISCUSSION Our data on the fatty acid composition o~ Euolena 9racilis, strain Z, grown in the dark on a complete medium (i.e. heterotrophically) confirm the presence of large concentrations of 20- and 22-carbon polyenoic acids. Docosa-4,7,10,13,16-pentaenoic acid represents a considerable proportion of the unsaturated fatty acids, and it can be labeled with 14C if the Euylena are grown in the presence of ~4C-acetate, 14C-linoleate or 14C-arachidonate. This polyene consistently contained the greatest proportion of the incorporated radioactivity from all three substrates. These studies do not permit us to specify the optimal conditions for incorporating radioactivity from these substrates into 22:5n-6 or for achieving maximal specific activity. Specific activities could be increased by using more labeled substrate or substrate of a higher specific activity (uniformly ~4C labeled as well as tritiated material). Chemical synthesis of the radioactive pentaene would yield high specific activities, but the difficulty of such synthesis precludes its use in most laboratories. The manner of biosynthesis of the 22:5n-6 was studied by chemical degradation of the hydrogenated derivative of the isolated, purified compound. Results of degradation of the polyene biosynthesized with l-[~4C]-linoleate or 1-[14C]-arachidonate as substrate clearly show that these were elongated rather directly to the 22-carbon derivative by a two-carbon fragment. The finding of minor amounts of ~4C in carbons other than the one represented by the original substrate indicates that a portion of the 14C-substrate was oxidized and the products of oxidation used in the elongation pathway. However, essentially no radioactivity was found in fatty acids of chain length shorter or more saturated than linoleic acid. Hulanicka et al. (1964) reported similar findings using ~4C-linoleate as substrate. However, they did not isolate or specifically identify 22:5n-6 in their studies. Results of the experiments utilizing 1-[~4C]acetate as substrate are more difficult to interpret. The ~4C-acetate was apparently used for synthesis of the whole chain, but the portion of the molecule near the methyl group was richer in 14C than the portion near the carboxyl group. Thus, it appears that a certain portion of the chain was synthesized rapidly while the specific activity of the acetyl CoA unit was high, and this portion was subsequently elongated
263
with carbons of decreasing specific activity. Since the substrate was labeled only in the carboxyl (No. 1) carbon, only odd numbered carbons were expected to be labeled. The amount of ~4C found in the evennumbered carbons in these experiments was too large to be explained as some artifact of the analysis. However, the exact mode by which ~4C entered these positions remains to be explained. Korn (1964) raised the possibility that the fatty acid composition of protists might be useful as a phylogenetic tool. Our studies indicate that these organisms may also be useful for the biosynthetic preparation of metabolically useful labeled polyenoic acids which are not available commercially and for which the chemical synthesis is not feasible in most laboratories. Acknowledyements--This study was supported in. part by U.S.P.H.S. Grants Nos. AM-06483 and HD-07694.
REFERENCES
BRIDGES R. B. & CONIGLIOJ. G. (1970) The metabolism of 4,7,10,13,16-[5-~4C]-docosapentaenoic acid in the testis of the rat. Biochim. biophys. Acta 218, 29-35. DAUBENW. G., HOERGERE. & PETERSENJ. W. (1953) Distribution of acetic acid carbon in high fatty acids synthesized from acetic acid by the intact mouse. J. Am. Chem. Soc. 75, 2347-2351. DAVISJ. T. & CONIGLIOJ. G. (1966) The biosynthesis of docosapentaenoic acid and other fatty acids by rat testes. J. biol. Chem. 241, 610-612. DAVIS J. T., BRIDGES R. B. & CONIGLIO J. G. (1966) Changes in lipid composition of the maturing rat testis. Biochem. J. 98, 342-346. FARQUHAR J. W., INSULL W. JR., ROSEN P., STOFFELW. & AHRENSE. H. (1959) The analysis of fatty acid mixtures by gas-liquid chromatography, Nutr. Rev. 17. (Supplement 2), 1-30. HULANICKAD., ERWINJ. & BLOCH K. (1964) Lipid metabolism of Euglena gracilis. J. biol. Chem. 239, 2778-2787. KORN E. D. (1964) The fatty acids of Euylena yracilis. J. Lipid Res. 5, 352-362. KUNAC WOLF-H. (1971) The total synthesis of polyynoic and polyenoic acids, with four, five, and six triple or double bonds. Hoppe-Seyler's Z. Physiol. Chem. 352, 542-548. METCALFEL. D. & SCHMITZA. A. (1961) The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33. 362-364.
BIOSYNTHESIS OF DOCOSA-4,7,10,13,16P E N T A E N O I C ACID IN E U G L E N A GRACILIS CLARENCE E, FOUCHE, JR.* AND JOHN G. CONIGLIO Department of Biochemistry, Vanderbilt University, Nashville, TN 37232, U.S.A.
(Received 21 September 1976) Abstract--1. Polyenoic acids, including arachidonic and docosa-4,7,10,13,16-pentaenoic (22: 5n-6) acids, comprise a large portion of the total fatty acids of Euglena gracilis grown in the dark. 2. 14C from 1-[14C]-acetate, 1-[14C]-linoleate, and 1-[~4C]-arachidonate was incorporated into 22:5n-6 in cultures grown in the dark in the presence of these suhstrates. 3. 1-[x4C]-linoleate and 1-[14C]-arachidonate were elongated and desaturated to 5-[14C]-22:5n-6 and 3-[~4C]-22:5n-6, respectively, with little reutilization of acetyl units produced by oxidation. 4. l-[14C]-acetate was utilized to form the entire chain of 22:5n-6, with the carbons nearer the methyl group having a richer isotope content than the carbons nearer the carboxyl group. 5. Some 14C from 1-[14C]-acetate apparently entered the even carbon positions of the polyene by some unexplained process. INTRODUCTION
the Results section). The labeled compound was introduced into 2.8 1. Fernbach culture flasks containing 1 1. of Euolena broth. The medium was then sterilized by autoclaving, and 1 ml of EucJlena culture was added aseptically. The cultures were kept in the dark by wrapping the flasks with aluminum foil and grown for 2 weeks at room temperature. They were harvested by centrifugation at 5000 rev/min for 10min in 500-ml polycarbonate bottles, resuspended in distilled water and re-centrifuged. The pellet was dispersed in a small quantity of distilled water and transferred to a 50-ml flask, in which it was hydrolyzed in 40~o ethanolic KOH (using hyroquinone as anti-oxidant) for 2 hr under a stream of nitrogen. After extraction of non-saponifiable material, the hydrolysate was acidified and the free fatty acids recovered by extraction with petroleum ether. Methyl esters were prepared using the method of Metcalfe & Schmitz (1961). 14C determinations were made by liquid scintillation spectrometry in BraSs solution or in a toluene solution containing diphenyloxazole. Quenching was checked and corrections made by the use of an internal 14C toluene standard. Analytical gas-liquid chromatography was done with a column of SP-2340 (10~o on 100/120 mesh Supelcoport, Supelco, Inc., Bellefonte, PA). Gas-liquid radiochromatography was done with a similar column, the effluent of which passed through a heated tube to a continuous flow, proportional detector. For preparative collection of the labeled polyene, a column provided with a 1:1 mixture of 10~o SP-2340 on 100/120 mesh Supelcoport and 15~o EGSS-X on 100/120 mesh Gas Chrom P (Applied Science Laboratories, State Park, PA) was used, and the effluent was trapped on silanized glass wool. The collected methyl esters were eluted from the glass wool with petroleum ether. Analytical ga~liquid chromatography and gas-liquid radiochromatography were used to check the chemical and radiopurity of the collected samples. These were > 95~o pure for all samples used for characterization or degradation. Location of the 14C in fatty acid molecule was done on the hydrogenated derivatives (Farquhar et al., 1959) by the procedure of Dauben et al. (1953).
Polyunsaturated fatty acid metabolism in rat testis has been a major area of investigation in this laboratory for many years. Of particular interest has been the finding that the concentration of one particular polyene, docosa-4,7,10,13,16-pentaenoic acid (22: 5n-6), increases dramatically at the time of sexual maturation (Davies et al., 1966). Past investigations have included the biosynthesis of this compound (Davis & Coniglio, 1966) as well as some brief studies of its metabolism (Bridges & Coniglio, 1970). The latter type of study is difficult due to the non-availability of the pure compound, either radioactive or nonradioactive. The chemical synthesis is rather formidable (Kunau, 1971). The presence of relatively large amounts of this polyene in Euglena gracilis (Korn, 1964; Hulanicka et al., 1964) led us to investigate its biosynthesis from labeled substrates. Our studies show that the lipids of the organism contain from 7 to 10~o of its total fatty acids as 22:5n-6 when it is grown in the dark. Furthermore, it incorporates 14C from 14C-acetate, 14C_linoleat e and 14C_arachidonate into 22: 5n-6 in significant quantities. Chemical degradation studies of the biosynthesized polyene were done to determine the manner of labeling.
MATERIALS AND METHODS l-[14C]-sodium acetate and 1-[~4C]-linoleic acid were obtained from New England Nuclear Corporation, Boston, MA and l-[14C]-arachidonic acid from Dhom Products, Inc., North Hollywood, CA. Euglena gracilis, strain Z, was obtained from the Carolina Biological Supply House, Burlington, NC. Stock cultures of the organism were maintained under continuous flourescent light in screw-cap culture tubes containing Difco Euolena broth. Separate cultures of Euglena were grown containing the ~4C-substrates (amount of radioactivity used is given in
RESULTS About 7~o of the total fatty acids of Euglena grown in the dark was 22:5n-6. Arachidonic acid was pres-
* Present Address: Veterans Administration Hospital, Nashville, Tennessee 37232, U.S.A. 261
262
Q~I.ARENCE E. I~'Ot;CHI: JR. AND JOHN G. ('ONIGI.RJ
Table 1. Incorporation of labeled substrates into total fatty acids of Eu~/le~, Total fatty acids ",, of substratc 1 4 ( '
Fatty acid Sodium 1-[14C]-acetate Sodium 1-[14C]-linoleate ~ Sodium l-[14C]-arachidonate 'l
2(1" 8 47
"
__.~-_~,n-( of substratc laC ,v, 32
:' average of 4 cultures. " average of 3 cultures. results available on only 1 culture. d average of 3 cultures.
ent at a level of about 10')o of the total fatty acids, 22:6n-3 at about 3~;; and 20:5n-3 at about 5'~0. Other polyenes of 18-, 20-, and 22-carbon chain length amounted to 15°~o of the total fatty acids. In Table 1 is shown the amount of incorporation of ~4C from various substrates into total fatty acids of Ewlena and the amount of incorporation specifically into 22:5n-6. The most efficient incorporation was obtained with l"*C-arachidonate (32'!;, of the added substratee was incorporated into the isolated 22:5n-6). The highest specific activity obtained with each of these substrates was 992 ~Ci/mmole from 1-[~'*C]-acetate of specific activity 2.0mCi/mmole: 3.8 #Ci/mmole from 1-[~4C]-linoleate of specific activity 56mCi/mmole and 221 iLCi/mmole from 1-[~4C]-arachidonate of specific acitivity 5 8 m C i / mmole. The ~4C-activity not found in 22:5n-6 in the cultures incubated with l'~C-acetate was distributed in a number of fatty acids, both saturated and unsaturated. However, in the cultures incubated with
~4C-linoleate. no ~4C was observed in fatty acids of carbon chain length less than 18, while in those cultures incubated with 14C-arachidonate, the 14C was almost entirely in 22:5n-6 (aside from that remaining in 20:4n-6). The distribution of the labeled carbon in the 22:5n-6 isolated from cultures incubated with l-[~4C]-arachidonate or with l-[14C]-linoleate is shown in Table 2. In the former very little ~4C was present in the carboxyl {No. 1) or 2 (No. 2) carbon, while the third carbon had essentially all the activity of the molecule. In the case of the l-[~C]-linoleate substrate the fifth carbon had most of the ~4C activity while very little was present in any of the other carbons. The use of l-[14C]-acetate as substrate gave a more complex picture. The results of these incubations are shown in Table 3. Generally, the odd numbered carbons had most of the ~~C (compared to the even-numbered carbons). It is also apparent that the amount of 14C increased toward the methyl end of the molecule contrasted with the carboxyl end.
Table 2. Dauben degradation of 22:5n-6 synthesized in the presence of sodium l-[~aC]-arachidonatc or 1-[l~C]-linoleate
Carbon 11o.
Substrate 1-[l~C]-arachidonate
1-[~4C]-linoleate
Specific activity of benzoic acid {counts/min mmolc ~1 [B]
Initial specific activity (counts/min mmole ~C) EA]
I 2 3 1 2 3 4 5
9562 4667 1900 2871 1874 956 38(1 140
930 1623 3(I.666 471 113 378 288 1748
Table 3. Dauben degradation of 22:5n-6 synthesized from 1-[~a(']-acetate Ratio: Carbon no. l 2 3 4 5 6 7
Specific activity carboxyl carbon Specific activity average carbon in chain
Exp. 1
Exp. 2
Exp. 3
Exp. 4
0.91 0.13 1.72
1.18 0.05 0.40 0.89
0.88 0.04 1.30 0.29 1.35
0.77 0.42 0.76 1.37 0.94 1.79 2.98
Ratio [B]/[A] 0.1 0.4 16.1 (1.2 0.1 (1.4 (1.8 12.5
Docosapentaenoic acid synthesis in Euglena oracilis DISCUSSION Our data on the fatty acid composition o~ Euolena 9racilis, strain Z, grown in the dark on a complete medium (i.e. heterotrophically) confirm the presence of large concentrations of 20- and 22-carbon polyenoic acids. Docosa-4,7,10,13,16-pentaenoic acid represents a considerable proportion of the unsaturated fatty acids, and it can be labeled with 14C if the Euylena are grown in the presence of ~4C-acetate, 14C-linoleate or 14C-arachidonate. This polyene consistently contained the greatest proportion of the incorporated radioactivity from all three substrates. These studies do not permit us to specify the optimal conditions for incorporating radioactivity from these substrates into 22:5n-6 or for achieving maximal specific activity. Specific activities could be increased by using more labeled substrate or substrate of a higher specific activity (uniformly ~4C labeled as well as tritiated material). Chemical synthesis of the radioactive pentaene would yield high specific activities, but the difficulty of such synthesis precludes its use in most laboratories. The manner of biosynthesis of the 22:5n-6 was studied by chemical degradation of the hydrogenated derivative of the isolated, purified compound. Results of degradation of the polyene biosynthesized with l-[~4C]-linoleate or 1-[14C]-arachidonate as substrate clearly show that these were elongated rather directly to the 22-carbon derivative by a two-carbon fragment. The finding of minor amounts of ~4C in carbons other than the one represented by the original substrate indicates that a portion of the 14C-substrate was oxidized and the products of oxidation used in the elongation pathway. However, essentially no radioactivity was found in fatty acids of chain length shorter or more saturated than linoleic acid. Hulanicka et al. (1964) reported similar findings using ~4C-linoleate as substrate. However, they did not isolate or specifically identify 22:5n-6 in their studies. Results of the experiments utilizing 1-[~4C]acetate as substrate are more difficult to interpret. The ~4C-acetate was apparently used for synthesis of the whole chain, but the portion of the molecule near the methyl group was richer in 14C than the portion near the carboxyl group. Thus, it appears that a certain portion of the chain was synthesized rapidly while the specific activity of the acetyl CoA unit was high, and this portion was subsequently elongated
263
with carbons of decreasing specific activity. Since the substrate was labeled only in the carboxyl (No. 1) carbon, only odd numbered carbons were expected to be labeled. The amount of ~4C found in the evennumbered carbons in these experiments was too large to be explained as some artifact of the analysis. However, the exact mode by which ~4C entered these positions remains to be explained. Korn (1964) raised the possibility that the fatty acid composition of protists might be useful as a phylogenetic tool. Our studies indicate that these organisms may also be useful for the biosynthetic preparation of metabolically useful labeled polyenoic acids which are not available commercially and for which the chemical synthesis is not feasible in most laboratories. Acknowledyements--This study was supported in. part by U.S.P.H.S. Grants Nos. AM-06483 and HD-07694.
REFERENCES
BRIDGES R. B. & CONIGLIOJ. G. (1970) The metabolism of 4,7,10,13,16-[5-~4C]-docosapentaenoic acid in the testis of the rat. Biochim. biophys. Acta 218, 29-35. DAUBENW. G., HOERGERE. & PETERSENJ. W. (1953) Distribution of acetic acid carbon in high fatty acids synthesized from acetic acid by the intact mouse. J. Am. Chem. Soc. 75, 2347-2351. DAVISJ. T. & CONIGLIOJ. G. (1966) The biosynthesis of docosapentaenoic acid and other fatty acids by rat testes. J. biol. Chem. 241, 610-612. DAVIS J. T., BRIDGES R. B. & CONIGLIO J. G. (1966) Changes in lipid composition of the maturing rat testis. Biochem. J. 98, 342-346. FARQUHAR J. W., INSULL W. JR., ROSEN P., STOFFELW. & AHRENSE. H. (1959) The analysis of fatty acid mixtures by gas-liquid chromatography, Nutr. Rev. 17. (Supplement 2), 1-30. HULANICKAD., ERWINJ. & BLOCH K. (1964) Lipid metabolism of Euglena gracilis. J. biol. Chem. 239, 2778-2787. KORN E. D. (1964) The fatty acids of Euylena yracilis. J. Lipid Res. 5, 352-362. KUNAC WOLF-H. (1971) The total synthesis of polyynoic and polyenoic acids, with four, five, and six triple or double bonds. Hoppe-Seyler's Z. Physiol. Chem. 352, 542-548. METCALFEL. D. & SCHMITZA. A. (1961) The rapid preparation of fatty acid esters for gas chromatographic analysis. Anal. Chem. 33. 362-364.