Identification of Branched Chain Fatty Acids in Baboon Liver Lipids A. Smith, A. G. Calder, E. Rona Morrsion and G. A. Garton Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, UK
A total of 26 monomethyl branched saturated fatty acids was identified in baboon liver lipids; these included a novel anteiso component with an odd number of carbon atoms in the chain (13-methylpentadecanoicacid).
INTRODUCTION
It was reported previously from this laboratory' that methyl branched fatty acids occurred in trace amounts in the liver lipids of the baboon (Papiocyanocephalus) and that, in animals depleted of vitamin BIZ, the amounts were somewhat enhanced, notably in baboons which had received injections of a biologically inactive analogue of the vitamin (cyanocobalamin monocarboxylic acid). Regardless of the vitamin B12 status of the animals, a similar range of branched chain fatty acids was apparently present, 14 of which were presumptively identified on the basis of their gas chromatographic properties. In this paper we report the identification, by gas chromatographic mass spectrometric (GCMS) analysis, of a total of 26 branched chain fatty acids (including a novel anteiso acid) in the liver lipids of three of the animals which were included in the above investigation. As already described,'.' one of these animals had been fed on a stock diet of fresh fruit, vegetables and high protein biscuits and the other two had received a synthetic diet with or without a supplement of vitamin B12;the baboon deprived of vitamin B12 had been given intramuscular injections of a B12 analogue (cyanocobalamin monocarboxylic acid) 15 months before it was killed. The proportion of branched chain components in the total fatty acids of the liver lipids of the baboon fed on the stock diet was 0.3%, for the animal given the synthetic diet plus vitamin B12 it was 0.6%, and for the vitamin B12-deprived animal it was 2.2%.
2-methyldodecanoate and methyl 2-methylheptadecanoate) were added to each preparation to avoid loss, at this stage of the analysis, of the very small amounts of branched chain esters derived from the baboon liver lipids. To facilitate subsequent analysis by GCMS, each of the three concentrates of branched chain esters was then subjected to preparative G C to eliminate almost entirely the carrier esters and any residual straight chain esters. The G C was effected at 190 "C in a 1.5 m glass column (internal diameter 4 mm) containing silane treated Celite (SO/lOO mesh) coated with 10% (w/w) Apiezon L grease; an effluent splitter (ratio 10 : 1) was employed and the branched chain esters were collected in a capillary tube cooled in a bath containing ethanc.1 and solid C02. These esters were then analysed by GCMS using a 1 0 0 m stainless steel, open tubular column (internal diameter 0.25 mm) coated with polymerized butanediol succinate and operated at 165 "C with helium as carrier gas at a flow rate of 1 ml min-I; the interface with the mass spectrometer (VG Micromass Model 16F) was an all glass, direct inlet system maintained at 250 "C. Mass spectra which were recorded at a scan speed of 1 s per decade either manually or with a twin floppy diskette data system were interpreted in the light of established ~ r i e r i a . " ~To establish unequivocally the identity of a component (13-methylpentadecanoic acid) which had not previously been reported as a naturally occurring acid, a reference sample was prepared chemically by oxidative degradation' of 14-methylhexadecanoic acid (Analabs Inc., USA). The mass spectrum of the authentic acid (Fig. 1) was identical in every respect with that of the acid present in baboon liver lipids. 100 100,
EXPERIMENTAL
80-
As in the previous investigation,' 50 g samples of formol preserved liver from each of the three baboons were taken for analysis. Total lipids extracted with chloroform+methanol ( 2 : 1, v/v) were saponified with excess 0.5 M ethanolic KOH and the resultant fatty acids converted to methyl esters by refluxing with methanol containing 1% (w/w) H2S04. The methyl esters were treated with mercuric acetate3 to remove most of the unsaturated components and then with urea4 to remove the bulk of the straight chain esters; before urea segregation carrier branched chain esters (methyl
60. 40 40. 20. 20 J a l 209 0
IIII I I I00
.JI/,,
I
t
I
I 1 I
200
150
t,
[M-311' [M-291'
I
!Id 250
10
m/r
Figure 1. Mass spectrum of the methyl ester of 13-methyl-
pentadecanoic acid. The arrows indicate components t o which reference is m ad e in the text.
CCC-0306-042X/79/0006-0345 $01.OO @ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 8, 1979 345
A. SMITH, A. G. CALDER, E. R MORRISON AND G. A. CARTON
RESULTS AND DISCUSSION The integrated ion current chromatograms obtained from each of the three preparations of branched chain esters were almost identical and it was thus evident that, as inferred from an earlier study,' the number and range of chain length of the components was essentially the same, regardless of the differences between the diets eaten by the three baboons. It is not possible from such chromatograms to determine the precise relative proportions of the fatty acid esters, though it was evident that anteiso acids and acids with a methyl substituent in position 4 were predominant components in each preparation. The total number of branched chain fatty acids which, as their methyl esters, were identified in the liver lipids of each animal are listed in Table 1, together with their equivalent chain length values. It can be seen that the acids include a number of members of several homologous series of monomethyl substituted acids which range in chain length from 12 to 17 carbon atoms, together with four components possessing an isopropyl group (is0 acids). The biosynthesis of methyl branched fatty acids, other than is0 acids, involves either acetyl-CoA or propionylCoA as primer unit and the utilization of methylmalonyl-CoA in place of malonyl-CoA at one or other step in the chain lengthening process; the methyl substituent thus appears on any even numbered carbon atom relative to that of the carboxyl group of the molecule. Is0 acids cannot arise in this way and their biosynthesis involves either isobutyryl-CoA or isovaleryl-CoA as primer unit for chain elongation with malonyl-CoA. All but one of the 26 methyl branched fatty acids which were identified in the baboon liver lipids have earlier been reported either as components of bacterial or avian (uropygial gland) lipids (see review^'^,'^) or of the subcutaneous triacylglycerols of barley fed lambs.I4 The exception is the anteiso acid (13-methylpentadecanoic acid) which, as noted above, has not been reported among naturally occurring anteiso acids; it is unusual in that it possesses an odd rather than an even number of carbon atoms in the chain. The mass spectrum of its methyl ester (Fig. 1) exhibits the following salient features: [MI" at m / z 270, base peak at m / z 74, [M-29]'>[M-31]' (a feature of the anteiso structures), and ketene and ketene-H20 peaks at m / z 209 and m / z 191 respectively indicative of a methyl substit-
Table 1. Identities and equivalent chain length values of branched chain fatty acid methyl esters derived from baboon liver lipids Identity of methyl ester
EquivalenT Chain, length valuea
12.70 12.80 13.38 13.52 13.80 14.28 14.33 14.36 14.41 14.53 14.70 14.80
10-Me-dodecanoate (anteiso) 2-Me-tridecanoate 4-Me-tridecanoate 12-Me-tridecanoate (iso) 2-Me-tetradecanoate 6-Me-tetradecanoate 8-Me-tetradecanoate 4-Me-tetradecanoate 10-Me-tetradecanoate 13-Me-tetradecanoate (iso) 12-Me-tetradecanoate (anteiso) 2-Me-pentadecanoate 6-Me-pentadecanoate 8-Me-pentadecanoate 4-Me-pentadecanoate 10-Me-pentadecanoate} 14-Me-pentadecanoate (iso) 13-Me-pentadecanoate (anteiso) 2-Me-hexadecanoate 6-Me-hexadecanoate 8-Me-hexadecanoate 10-Me-hexadecanoate 4-Me-hexadecanoate 12-Me-hexadecanoate 15-Me-hexadecanoate (iso) 14-Me-hexadecanoate (anteiso)
}
15.29 15.36 15.54 15.70 15.80
1
a
16.29 16.32 16.37 16.42 16.54 16.70
Liquid phase: polymerized butanediol succinate.
uent on carbon 13; the intensities of these two peaks are c. 4% of the base peak. Further studies in this laboratory (unpublished) on the methyl branched fatty acids of the triacylglycerols of barley fed lambs have revealed that this anteiso acid is among the many components which were not previously identified.14 It seems possible that it could originate as a result of a-oxidation of the anteiso acid, 14-methylhexadecanoic acid which, as Table 1 shows, is also one of the component branched chain acids present in baboon liver lipids.
Acknowledgement E. Rona Morrison took part in this work during the tenure of Research Training Scholarship awarded by the Wellcome Trust.
a
REFERENCES 1. G. A. Garton, J. R. Scaife, A. Smith and R. C. Siddons, Lipids 10, 855 (1975). 2. R. C. Siddons, Nature (London)247, 308 (1974). 3. E. Jantzen and H. Andreas, Angew. Chern. 70, 656 (1978). 4. R. G. Ackman and S.N. Hooper, Comp. Biochem. Physiol. 24, 549 ( 1968). 5. S. Abrahamsson, S. Stallberg-Stenhagen and E. Stenhagen, in Progress in the Chemistry of Fats and Other Lipids. Vol. 7, ed. by R. T. Holman and T. Malkin, p. 1. Pergamon Press, Oxford (1963). 6. J. M. 6. Apon and N. Nicolaides, J. Chromatogr. Sci. 13,467 (1975). 7. K. E. Murray, Aust. J. Chem. 12, 657 (1959). 8. J. R. Scaife and G. A. Garton, Biochern. SOC.Trans. 3, 993 (1975).
346 BIOMEDICAL MASS SPECTROMETRY, VOL. 6,
NO. 8, 1979
9. J. S. Buckner and P. E. Kolattukudy, Biochemistry 14, 1774 (1975). 10. J. R. Scaife, K. W. J. Wahle and G. A. Garton, Biochem. J. 176, 799 (1978). 11. M. G. Horning, D. 6. Martin, A. Karmen and P. R. Vagelos, J. Biol. Chem. 236,669 (1961). 12. A. K. Sen Gupta, Fette Seifen Anstrichm. 74, 693 (1972). 13. G. A. Garton, Rep. Roweff lnst. 31, 124 (1975). 14. W. R. H. Duncan, A. K. Lough, G. A. Garton and P. Brooks, Lipids 9, 669 (1974).
Received 29 January 1979 @ Heyden & Son Ltd, 1979
@ Heyden & Son Ltd, 1979
A total of 26 monomethyl branched saturated fatty acids was identified in baboon liver lipids; these included a novel anteiso component with an odd number of carbon atoms in the chain (13-methylpentadecanoicacid).
INTRODUCTION
It was reported previously from this laboratory' that methyl branched fatty acids occurred in trace amounts in the liver lipids of the baboon (Papiocyanocephalus) and that, in animals depleted of vitamin BIZ, the amounts were somewhat enhanced, notably in baboons which had received injections of a biologically inactive analogue of the vitamin (cyanocobalamin monocarboxylic acid). Regardless of the vitamin B12 status of the animals, a similar range of branched chain fatty acids was apparently present, 14 of which were presumptively identified on the basis of their gas chromatographic properties. In this paper we report the identification, by gas chromatographic mass spectrometric (GCMS) analysis, of a total of 26 branched chain fatty acids (including a novel anteiso acid) in the liver lipids of three of the animals which were included in the above investigation. As already described,'.' one of these animals had been fed on a stock diet of fresh fruit, vegetables and high protein biscuits and the other two had received a synthetic diet with or without a supplement of vitamin B12;the baboon deprived of vitamin B12 had been given intramuscular injections of a B12 analogue (cyanocobalamin monocarboxylic acid) 15 months before it was killed. The proportion of branched chain components in the total fatty acids of the liver lipids of the baboon fed on the stock diet was 0.3%, for the animal given the synthetic diet plus vitamin B12 it was 0.6%, and for the vitamin B12-deprived animal it was 2.2%.
2-methyldodecanoate and methyl 2-methylheptadecanoate) were added to each preparation to avoid loss, at this stage of the analysis, of the very small amounts of branched chain esters derived from the baboon liver lipids. To facilitate subsequent analysis by GCMS, each of the three concentrates of branched chain esters was then subjected to preparative G C to eliminate almost entirely the carrier esters and any residual straight chain esters. The G C was effected at 190 "C in a 1.5 m glass column (internal diameter 4 mm) containing silane treated Celite (SO/lOO mesh) coated with 10% (w/w) Apiezon L grease; an effluent splitter (ratio 10 : 1) was employed and the branched chain esters were collected in a capillary tube cooled in a bath containing ethanc.1 and solid C02. These esters were then analysed by GCMS using a 1 0 0 m stainless steel, open tubular column (internal diameter 0.25 mm) coated with polymerized butanediol succinate and operated at 165 "C with helium as carrier gas at a flow rate of 1 ml min-I; the interface with the mass spectrometer (VG Micromass Model 16F) was an all glass, direct inlet system maintained at 250 "C. Mass spectra which were recorded at a scan speed of 1 s per decade either manually or with a twin floppy diskette data system were interpreted in the light of established ~ r i e r i a . " ~To establish unequivocally the identity of a component (13-methylpentadecanoic acid) which had not previously been reported as a naturally occurring acid, a reference sample was prepared chemically by oxidative degradation' of 14-methylhexadecanoic acid (Analabs Inc., USA). The mass spectrum of the authentic acid (Fig. 1) was identical in every respect with that of the acid present in baboon liver lipids. 100 100,
EXPERIMENTAL
80-
As in the previous investigation,' 50 g samples of formol preserved liver from each of the three baboons were taken for analysis. Total lipids extracted with chloroform+methanol ( 2 : 1, v/v) were saponified with excess 0.5 M ethanolic KOH and the resultant fatty acids converted to methyl esters by refluxing with methanol containing 1% (w/w) H2S04. The methyl esters were treated with mercuric acetate3 to remove most of the unsaturated components and then with urea4 to remove the bulk of the straight chain esters; before urea segregation carrier branched chain esters (methyl
60. 40 40. 20. 20 J a l 209 0
IIII I I I00
.JI/,,
I
t
I
I 1 I
200
150
t,
[M-311' [M-291'
I
!Id 250
10
m/r
Figure 1. Mass spectrum of the methyl ester of 13-methyl-
pentadecanoic acid. The arrows indicate components t o which reference is m ad e in the text.
CCC-0306-042X/79/0006-0345 $01.OO @ Heyden & Son Ltd, 1979
BIOMEDICAL MASS SPECTROMETRY, VOL. 6, NO. 8, 1979 345
A. SMITH, A. G. CALDER, E. R MORRISON AND G. A. CARTON
RESULTS AND DISCUSSION The integrated ion current chromatograms obtained from each of the three preparations of branched chain esters were almost identical and it was thus evident that, as inferred from an earlier study,' the number and range of chain length of the components was essentially the same, regardless of the differences between the diets eaten by the three baboons. It is not possible from such chromatograms to determine the precise relative proportions of the fatty acid esters, though it was evident that anteiso acids and acids with a methyl substituent in position 4 were predominant components in each preparation. The total number of branched chain fatty acids which, as their methyl esters, were identified in the liver lipids of each animal are listed in Table 1, together with their equivalent chain length values. It can be seen that the acids include a number of members of several homologous series of monomethyl substituted acids which range in chain length from 12 to 17 carbon atoms, together with four components possessing an isopropyl group (is0 acids). The biosynthesis of methyl branched fatty acids, other than is0 acids, involves either acetyl-CoA or propionylCoA as primer unit and the utilization of methylmalonyl-CoA in place of malonyl-CoA at one or other step in the chain lengthening process; the methyl substituent thus appears on any even numbered carbon atom relative to that of the carboxyl group of the molecule. Is0 acids cannot arise in this way and their biosynthesis involves either isobutyryl-CoA or isovaleryl-CoA as primer unit for chain elongation with malonyl-CoA. All but one of the 26 methyl branched fatty acids which were identified in the baboon liver lipids have earlier been reported either as components of bacterial or avian (uropygial gland) lipids (see review^'^,'^) or of the subcutaneous triacylglycerols of barley fed lambs.I4 The exception is the anteiso acid (13-methylpentadecanoic acid) which, as noted above, has not been reported among naturally occurring anteiso acids; it is unusual in that it possesses an odd rather than an even number of carbon atoms in the chain. The mass spectrum of its methyl ester (Fig. 1) exhibits the following salient features: [MI" at m / z 270, base peak at m / z 74, [M-29]'>[M-31]' (a feature of the anteiso structures), and ketene and ketene-H20 peaks at m / z 209 and m / z 191 respectively indicative of a methyl substit-
Table 1. Identities and equivalent chain length values of branched chain fatty acid methyl esters derived from baboon liver lipids Identity of methyl ester
EquivalenT Chain, length valuea
12.70 12.80 13.38 13.52 13.80 14.28 14.33 14.36 14.41 14.53 14.70 14.80
10-Me-dodecanoate (anteiso) 2-Me-tridecanoate 4-Me-tridecanoate 12-Me-tridecanoate (iso) 2-Me-tetradecanoate 6-Me-tetradecanoate 8-Me-tetradecanoate 4-Me-tetradecanoate 10-Me-tetradecanoate 13-Me-tetradecanoate (iso) 12-Me-tetradecanoate (anteiso) 2-Me-pentadecanoate 6-Me-pentadecanoate 8-Me-pentadecanoate 4-Me-pentadecanoate 10-Me-pentadecanoate} 14-Me-pentadecanoate (iso) 13-Me-pentadecanoate (anteiso) 2-Me-hexadecanoate 6-Me-hexadecanoate 8-Me-hexadecanoate 10-Me-hexadecanoate 4-Me-hexadecanoate 12-Me-hexadecanoate 15-Me-hexadecanoate (iso) 14-Me-hexadecanoate (anteiso)
}
15.29 15.36 15.54 15.70 15.80
1
a
16.29 16.32 16.37 16.42 16.54 16.70
Liquid phase: polymerized butanediol succinate.
uent on carbon 13; the intensities of these two peaks are c. 4% of the base peak. Further studies in this laboratory (unpublished) on the methyl branched fatty acids of the triacylglycerols of barley fed lambs have revealed that this anteiso acid is among the many components which were not previously identified.14 It seems possible that it could originate as a result of a-oxidation of the anteiso acid, 14-methylhexadecanoic acid which, as Table 1 shows, is also one of the component branched chain acids present in baboon liver lipids.
Acknowledgement E. Rona Morrison took part in this work during the tenure of Research Training Scholarship awarded by the Wellcome Trust.
a
REFERENCES 1. G. A. Garton, J. R. Scaife, A. Smith and R. C. Siddons, Lipids 10, 855 (1975). 2. R. C. Siddons, Nature (London)247, 308 (1974). 3. E. Jantzen and H. Andreas, Angew. Chern. 70, 656 (1978). 4. R. G. Ackman and S.N. Hooper, Comp. Biochem. Physiol. 24, 549 ( 1968). 5. S. Abrahamsson, S. Stallberg-Stenhagen and E. Stenhagen, in Progress in the Chemistry of Fats and Other Lipids. Vol. 7, ed. by R. T. Holman and T. Malkin, p. 1. Pergamon Press, Oxford (1963). 6. J. M. 6. Apon and N. Nicolaides, J. Chromatogr. Sci. 13,467 (1975). 7. K. E. Murray, Aust. J. Chem. 12, 657 (1959). 8. J. R. Scaife and G. A. Garton, Biochern. SOC.Trans. 3, 993 (1975).
346 BIOMEDICAL MASS SPECTROMETRY, VOL. 6,
NO. 8, 1979
9. J. S. Buckner and P. E. Kolattukudy, Biochemistry 14, 1774 (1975). 10. J. R. Scaife, K. W. J. Wahle and G. A. Garton, Biochem. J. 176, 799 (1978). 11. M. G. Horning, D. 6. Martin, A. Karmen and P. R. Vagelos, J. Biol. Chem. 236,669 (1961). 12. A. K. Sen Gupta, Fette Seifen Anstrichm. 74, 693 (1972). 13. G. A. Garton, Rep. Roweff lnst. 31, 124 (1975). 14. W. R. H. Duncan, A. K. Lough, G. A. Garton and P. Brooks, Lipids 9, 669 (1974).
Received 29 January 1979 @ Heyden & Son Ltd, 1979
@ Heyden & Son Ltd, 1979
