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High resolution NMR studies of fish oils
Chemistry and Physics of Lipids, 59 ( 1991) 83-89 Elsevier Scientific Publishers Ireland Ltd.
83
High resolution NMR studies of fish oils F.D. Gunstone Chemistry Department, The University, St Andrews. F(l'e. KYI6 9ST (U.K.)
(Received April 12th, 1991; revision received May 28th, 1991; accepted May 28th, 1991) The high resolution NMR spectra of four fish oils have been recorded. Signals in the 13C-NMRspectra are assigned and attention is drawn to specific signals for the n-3 group of acids and also individually for DHA, EPA and stearidonic acid. Keywords: high resolution NMR spectra; tH; t3C; fish oils; n-3 acids; EPA: DHA; stearidonic acid.
Introduction High resolution N M R spectroscopy is used increasingly as a technique to provide insight into the nature of the mixtures present in natural oils and fats and other lipids. This applies particularly to 13C-NMR spectra. For example, Wollenberg [1] demonstrated that the chemical shifts for signals associated with the acyl carbon atoms (C 1) depend on whether the chain is ct or/3 linked to glycerol and on the nature of the unsaturation in the chain, if any. The signals for the olefinic carbon atoms also depend on whether they are present in ~ or /~ chains. Wollenberg exploited this observation by determining the distribution of saturated and unsaturated acids between oL and/~ chains. However, to get satisfactory quantitative results relaxation times had to be included in the protocol for collecting spectra and this led to data collection over 5 h or more. We came to similar conclusions from our examination of a range of synthetic glycerides alone and as simple mixtures [2]. Our spectra were obtained without relaxation periods in about 30 rain. Realising that the small changes in chemical shift resulting from unsaturation at position 9 are larger Correspondence to: F.D. Gunstone, Chemistry Department, The University, St Andrews, Fife, KYI6 9ST, U.K. Abbreviations: DHA, docosahexaenoic acid: DPA, docosapentaenoic acid; EPA (e)icosapentaenoic acid.
when the double bond is closer to the acyl group we examined seed oils containing petroselinic acid (18:1 6c) [3] and ~-linolenic acid (18:3 6c9c 12c) [41. The results were encouraging in that we could determine the amounts of these acids and their distribution between the o~ and /3 positions from our 30-min spectra. Other oils with unsaturation close to the carboxyl group include the fish oils which contain important n-3 polyene acids such as D H A (22:6 4,7,10,13,16,19), DPA (22:5 7,10,13,16,19), EPA (20:5 5,8,11,14,17), and stearidonic acid (18:4 6,9,12,15) and we now report on our spectroscopic examination of four fish oils (see Experimental). Fish oils contain complex mixtures of fatty acids and as a preliminary to this investigation we previously examined six pure n-3 polyene esters [5]. In Table I we summarise some important data (slightly revised and extended) from our earlier paper which will be used in the subsequent discussion. We will draw attention to several signals which are specific for individual acids and for groups of related acids. These may be of value in the spectroscopic examination of fish oils but improved quantitative results will require spectra collected more slowly or with the addition of relaxation agents. Our assignments are based on information taken from our previous papers and from our knowledge of the composition of the four fish oils determined by gas chromatography (Table II).
0009-3084/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
84 TABLE 1 13C-NMR data for six n-3 polyene methyl esters adapted from Ref. 5.
CI C2 C3 C4 C5 C6 C7 o~l Allylic o~2 Other Double allylic C6 o~5 Olefinic d ~03 t,,'4
22:6 (A4...)a
20:5 (A5...)a
18:4 (A6...)a
22:5 (A7...)a
20:4 (A8...)a
18:3 (A9...)a
173.46" 34.01 (22.81 )b . . 14.28
173.99 33.43* 24.80 (26.57) b . 14.29
174.03 33.97 24.60* 29.12 (26.88) b -
174.14 34.05 24.88 28.81" 29.27 (27.06) b
14.28
14.27
174.22 34.08 24.93 29.07 28.92* 29.44 (27.18) b 14.29
174.15 34.08 24.99 29.23 29.18 29.15 29.63 14.31
.
20.57 22.81(C3)
20.58 26.57(C4)
20.58 26.88(C5)
20.57 27.06(C6)
20.58 27.18(C7)
20.61 27.25(C8)
25.65 (3) c 25.59 25.56
25.64 (3) c
25.65 (2) c
25.66 (3) c
25.65 (2) c
25.67
25.56
25.56
25.56
25.56
25.57
132.02 127.03
131.99 127.05
132.02 127.06
132.00 127.04
131.88 127.17
132.00 127.02
alndicates the position of the first double bond with respect to the ester group. bThis signal is also allylic. Clndicates the number of carbon atoms represented by this signal. dUnassigned olefinic signals lying between the o~4 and oJ3 values are listed in Ref. 5. *This chemical shift is markedly different from others in the horizontal row. It is 3' to a double bond.
T A B L E I1 Major component acids for four fish oils. G C analysis on a megabore column (DB-23, J and W Scientific) using helium as carrier gas. CPL a
BT a
MAX a
CLO a
Saturated Monoene Major n-3 acids 18:4 20:5 (EPA) 22:5 (DPA) 22:6 ( D H A )
24.2 25.7
27.2 23.7
25,6 25,3
16.5 49.0
3.7 16.3 2.6 12.4
3.2 15.8 2.8 9.4
1.9 17.4 2.9 11.4
2.7 8.5 1.4 I 1.9
EPA ÷ D H A EPA + D H A
28.7 1.31
25.2 1.68
28.8 1.53
20.4 0.71
aSee Experimental.
85
Experimental The fish oils examined were: (i) CPL fish oil (provided by Lipid Teknik, Stockholm), (ii) Fish oil for Biomedical Tests provided by the National Institute of Health (U.S.A.), (iii) Max EPA, and (iv) cod liver oil (purchased locally). On the basis of gas chromatography of their methyl esters these oils contained the major component acids listed in Table II. EPA and DHA together range from 20% to 29'7,, and the ratio of EPA to DHA from 0.7 to 1.7. Cod liver oil shows the greatest difference from the remainder in its high level of monoene acids and reduced proportions of saturated and polyene acids. The spectra were obtained using CDCI3 solutions and a Brucker AM 300 spectrometer. Data were acquired over 20,000 Hz into 32K data points and zero-filled to 64K. When necessary, resolution enhancement was increased using Gaussian multiplication [6].
Results and Discussion tH-NMR spectra The fish oils show the expected range of signals in their ~H-NMR spectra but with some additional information. In addition to the usual CH3 signal at 0.85 ppm there is a triplet at 0.95 ppm arising from all the n-3 acids. Additional signals of slightly higher chemical shift also appear on the side of signals at 2.35 (C2), 2.1 (allylic), and 1.65 ppm (C3). The first of these is a signal assigned to C3 of D H A which is also an allylic carbon atom [5], the others arise from allylic or C3 hydrogens under the additional influence of a nearby double bond in the polyene acids. ~3C-NMR spectra The assignments which follow are based on our study of several vegetable oils (Gunstone, unpublished results) and on previously published information [2] including our paper on n-3 polyene esters [5]. We have abstracted some of the more important data from this paper in Table I and have there drawn attention to specific signals characteristic of each of the major polyene acids.
Glycerol, CI, C2 and C3 carbon atoms (Table III) Most vegetable oils containing only saturated, oleic, linoleic (and possibly linolenic) acids show two signals for the glycerol carbon atoms at about 68.9 (O) and 62.1 ppm (c~) with the latter twice the size of the former because it represents two carbon atoms. These are also apparent as the largest signals in the fish oils but they are accompanied by smaller signals of higher chemical shift. These probably arise from n-3 polyene acids in the B chain but we cannot be more specific. CI signals usually occur at about 173.2 (ct) and 172.8 ppm (3) with a difference of about 0.40. These are present in the fish oils and are accompanied by other signals associated with the ct chains and the B chain. On the basis of our results with methyl esters summarised in Table I we consider that the signals at 172.8 and 172.4-172.5 (difference 0.37) are due to EPA in the ct and B positions, respectively. The signal at 172.0 probably results from DHA in the B position whilst that from DHA in the c~-position (if any) would be expected around 172.4 and may overlap with EPA (3). The C2 signals for c~ and 3 chains are fairly close together (separation about 0.17 ppm) with chemical shifts about 34.2 and 34.05 ppm. We observe up to three further signals and consider that those around 33.5-33.6 and 33.3-33.4 (difference 0.18) are related to EPA in the 3 and ct position, respectively. This carbon atom is 3' to the double bond in the 5-position of EPA and is expected to be about 0.5-0.6 ppm lower than similar C2 atoms not influenced by a nearby double bond. C3 carbon atoms give two signals around 24.9 ppm (difference 0.04) for the oe and 3 chains but these two signals usually appear only after resolution enhancement. We observe three or four signals for this carbon atom. That with the highest chemical shift is the usual value corresponding to most of the acyi chains in both o~ and # positions. The remaining signals are probably due to n-3 acids. DHA will not give a signal in this region because C3 is also allylic and its chemical shift will be lower. It appears in the same range as the ¢02 carbon atoms and will be discussed further with that group of signals. Bearing in mind that EPA is the n-3 acid present in highest proportion and the
86 T A B L E I11 C h e m i c a l shifts ( p p m ) a n d intensities f o r glycerol, C I , C 2 a n d C3 c a r b o n a t o m s . CPL a Glycerol
CI
69.16 69.06
0.8 0.9
68.94 62.07
2.3 5.2
68.94 62.09
2.5 5.4
173.01
2.6
173.12
1.0 0.9
172.93 172.88 172.73 172.62
0.5
172.55 172.51 172.41 172.04
0.4 0.5 0.3 0.6
172.05
0.5
34.18 34.02 33.89 33.53 33.34
2.6 8.3 1.5 0.9 2.0
34.21 34.04 33.91 33.57 33.38
2.9 8.2 1.6 0.9 2.1
34.24 34.08 33.95 33.62 33.44
2.8 7.0 1.2 0.5 2.2
24.90 24.78
9.1 1.8
24.89 24.78
8.7 1.9
24.92
24.72 24.49
2.9 1.2
24.72 24.49
2.5 1.4
24.76 24.52
0.4
69.23
CLO a
1.2 0.9
172.43 171.96
C3
MAX a
69.16 69.06
172.80 172.63 -
C2
BT a
0.8
-
Ass. b
69.23
1.4
-
69.01 62. I 1
3.4 7.4
/3 t~
4.6
ct
69.00 62.14
2.4 4.9
3.5
173.14
2.5
173.02
0.5 1.1 1.5 0.2
172.89 172.75 -
0.9 1.3
172.64 -
1.8
171.97
0.9
34.21 34.05
3.7 10.8
33.58 33.40
0.9 1.3
7.5
24.91 24.82
12.3 2.4
2.8 1.0
24.75 24.52
2.0 0.9
-
-
E P A c~ /~ EPA D H A /~ /~ ct EPA/3 E P A ct /~ c~ D P A etc. EPA 18:4
aSee E x p e r i m e n t a l . b S o m e o f these a s s i g n m e n t s a r e o n l y t e n t a t i v e (see text),
relative values of the chemical shifts for the methyl esters (Table I) we have assigned the signals as follows: 24.5, 18:4 (stearidonic acid); 24.75, EPA; 24.8, DPA and possibly 20:4 and 22:4.
o~1, o~2 and oJ3 signals (Table IV) The two 601 signals correspond to n-3 (14.3 ppm) and n-9/saturated (14.2 ppm). These oils contain only low levels of n-6 polyene acids and since the signal for this group is not well separated from that for the n-9/saturated chain (difference about 0.04) it is not apparent in these spectra. The major o~2 signal is derived from n-9/saturated acids. One oil shows a signal for n-6 acids. The o~2 atom in the important n-3 acids is also allylic and is discussed along with that group of
signals. The signal at 22.8 ppm observed with one oil may be due to DHA C3 which is influenced by its position with respect to the ester group and the adjacent double bond. The o~3 atom for n-3 acids is olefinic (see appropriate discussion). A small signal for n-6 acids is apparent in three of the spectra. The major signal expected at 31.9-32.0 (n-9 and saturated acids) is accompanied by a smaller but significant signal about 31.85 ppm but we have no explanation of this at the present time.
Allylic signals (Table IV) Allylic signals are easily divided into two categories depending on whether the carbon atoms are adjacent to one or two double bonds. These
87
TABLE Chemical
IV s h i f t s ( p p m ) a n d i n t e n s i f i e s f o r o~1, oJ2, oJ3 a n d a l l y l i c c a r b o n
CPL a
¢01
w 2
CLO a
Ass. b
14.30
5.5
14.29
4.3
14.26
3.5
14.28
4.6
10.2
14.13
9.1
14.10
7.9
14.11
13.7
-
13.81
0.6
-
-
-
22.92
0.2
-
-
-
22.81
1.0
-
-
22.72
9.8
22.72
22.62 32.83
1.0 0.2
-
10.6
-
Allylic
MAX a
14.14
22.74 w3
BT a
atoms.
9.2
32.19
0.2
-
9.4 2.7
31.97 31.83
7.5 3.3
31.97 31.84
7.2 2.1
31.57
0.9
31.56
0.9
31.57
0.7
30.02 -
0.4
. 27.47
0.2
27.41
0.4
27.21
8.7 6.2
27.25 27.21
7.4 5.6
27.27 27.22
27.07
0.6
27.06
0.7
26.95
0.4
26.95
26.88
1.5
26.53
3.1
27.25
DHA 15.6
31.97 31.85
11.8 1 3.7 J
n-9, s a t
-
n-6
-
-
-
-
-
.
.
C3
n-9, s a t n°6 -
.
6.7 4.5
-
27.27 -
17.21
-
27.05
1.0
AIlylic
0.3
-
-
26.87
1.6
26.89
1.2
26.89
1.2
18:4 ( C 5 )
26.53
2.7
26.57
2.6
26.56
2.0
EPA (C4)
-
J DPA (C6)
25.94
0.3
25.67
21.2
25.67
18.3
25.70
14.6
25.70
17.3
25.58
7.5
25.58
6.1
25.61
4.9
25.60
6.6
-
n-9, s a t
-
31.99 31.85
-
22.73
n-3
-
]Double B Lallylic ['DHA
[,o5
C 6 n-3
B
-
25.38
0.3
-
-
-
25.13
0.2
-
-
-
-
25.10
0.2
-
-
-
20.59
4.0
20.60
20.59
4.8
aSee Experimental. bSome of these assignments
3.1
20.60
-
4.0
n-3 w2
a r e o n l y t e n t a t i v e ( s e e text).
signals usually occur at about 27.2-27.3 (sometimes appearing as two signals) and 25.6-25.7 ppm. In these fish oils the significant signals at about 27.25, 27.20 and 25.70 ppm correspond to these major allylic signals. In addition the signal of 20.6 ppm derives from the w2 carbon atom in all n-3 acids. There remain several signals for which we make the following suggestions: 25.6 ppm, DHA C6 and w5 carbon atoms in n-3 acids; 26.5-26.6 ppm, EPA C4; 26.9 ppm, stearidonic acid C5; 26.95-27.1 ppm, DPA C6 and other n-3 acids. The allylic carbon atoms at C6 (DHA), C4
(EPA), and C5 (stearidonic acid) come under the additional influence of the ester group and have lower chemical shifts than when this influence is absent. As already indicated the C3 (DHA) signal overlaps with the w2 signals.
Olefinic signals (Table V) Each of the fish oils gives 15-20 olefinic signals (before resolution enhancement) and this has proved to be the most difficult part of the spectrum to assign [5]. For the sake of completeness we present all our results in Table V but we are able to make
88 TABLE V Chemical shifts (ppm) and intensities for olefinic carbon atoms. CPL a 131.92
BT a 4.0
-
130.06 129.95 129.88 129.78 129.67 129.48 129.45 128.99 128.77 128.53 128.25 128.14 128.06 127.98 127.86 127.67 127.59 127.06
0.6 3.7 2.3 1.8 3.4 2.0 2.1 3.4 3.4 5.0 8.5 5.7 7.6 3.9 5.9 2.5 0.6 4.8
131.97 130,42 130.27 130,11 129.98 129.91 129.81 129.69 129.51 129.47 129.10 129.00 128.78 128.56 128.41 128.27 128.16 128.08 127.99 127.88 127.67 . 127.06
MAX a 3.9 0.3 0.2 0.8 4.2 1.6 1.3 3.4 1.5 1.7 0.2 3.0 3.0 4.4 1.3 7.0 4.8 6.1 2.6 4.6 1.6 . 4,1
132.02 130.02 129.94 129.84 129.72 129.51 129.03 128.81 128.61 128.32 128.24 128.20 128.12 128.02 127.92 127.70 . 127.10
CLO a 3.3
131.96 129.99 129.91 129.80 129.70 129.49 129.03 128.79 128.57 128.29 128.17 128.10 128.00 127.90 127.70
3.3 1.7 1.4 2.9 1.5
2.6 2.7 3.7 6.2 3.4 4.1 5.6 2.3 4.0 1.6
Ass.b 4.2
6.2 5.3] 4.9 J 5.9 2.5
2.3 2.3 4.5 7.7 4.0 7.0 3.6 4.7 2.6
n-3 ¢~3 OI0 Monoene 09 -
. 3.3
127.09 4.2
n-3
aSee Experimental. bSome of these assignments are only tentative (see text). O = oleic (and other A9 acids).
only a limited number of assignments. The signals of highest (131.9-132.0 ppm) and lowest (127.05-127.1 ppm) chemical shift are associated w i t h t h e oJ3 a n d oJ4 c a r b o n a t o m s r e s p e c t i v e l y in all t h e n-3 p o l y e n e a c i d s . S i g n a l s a t 1 2 9 . 9 5 - 1 3 0 . 0 p p m and 129.65-129.7 ppm arise from CI0 and C9 r e s p e c t i v e l y in o l e i c a c i d a n d o t h e r A 9 m o n o e n e s . W e f u r t h e r b e l i e v e t h a t t h e s i g n a l s a t 129.9 a n d 129.8 p p m c o m e f r o m o t h e r m o n o e n e a c i d s w i t h unsaturation further from the carboxyl group than A9. T h e h i g h e r i n t e n s i t i e s o f t h e s e f o u r m o n o e n e s i g n a l s i n c o d l i v e r oil is c o n s i s t e n t w i t h t h e h i g h level o f m o n o e n e a c i d s i n t h a t oil ( T a b l e 1I).
General comments on assignments In Table VI we compare some results for methyl e s t e r s w i t h t h o s e f o r g l y c e r o l e s t e r s in r e s p e c t o f c a r b o n a t o m s 2 a n d 3. T h e d i f f e r e n c e s in c h e m i c a l s h i f t s f o r t h e s e t w o t y p e s o f e s t e r s a r e s m a l l a n d we feel j u s t i f i e d in a p p l y i n g d a t a a v a i l a b l e f o r m e t h y l esters for assignments of glycerol ester spectra. A l s o , in a n y g r o u p o f s i g n a l s f o r a c a r b o n a t o m we compare the major chemical shift (for saturated a n d A9 o r n - 9 acyl c h a i n s ) w i t h t h a t o f s m a l l e r signals in the same region. A reduction of -0.4-0.5 p p m i n d i c a t e s t h a t t h e c a r b o n a t o m is 7 t o d o u b l e b o n d . T h i s l o w e r v a l u e is a p p a r e n t , f o r
89 TABLE VI Chemical shifts (ppm) for C2 and C3 atoms in methyl and glycerol esters. C2 Methyl ester Saturated C13-C21 a CI2, CI4, CI6 b Oleic b Linoleic b 18:1 (6) b'c 18:3 (6,9,12) b'd
34.18 34.1 ! 34.10 34.01 33.97
C3 Giyceride
34.24, 34.20, 34.19, 34.11, 34.06,
34.67 34.04 34.01 33.95 33.91
Methyl ester
25.11 24.98 24.94 24.63 24.63
Glyceride
24.92 24.87 24.85 24.53 24.50
aRef. 7. bUnpublished information (F.D.G.). CRef. 3. dRef. 4.
example, at C1, for DHA ( A 4 . . . ) , C2 for EPA (A5...), and C3 for stearidonic acid (A6...). Conclusion To exploit the 13C-NMR spectra of fish oils we need (a) to assign all or most of the chemical shifts with a view to identifying those which are specific for one fatty acid or for a group of acids and (b) to improve the quantitative aspects of the spectra. This paper is devoted to the assignments: most of these are quite firm, but some are only tentative, and some have yet to be made. Finally we list those signals which provide spectroscopic information about the n-3 acids as a group or about individual members of that group. n-3 acids: DHA: EPA: Stearidonic:
~01 and olefinic signals CI(#) and C3 (in the oJ2 group) CI (a and #), C2 (c~ and ~/), C3, and ailylic (C4) C3 and allylic (C5)
It is clear that the t3C-NMR spectra of fish oils
contain useful information relating to the important n-3 acids, both as a group and individually. With improved quantitation 13C-NMR spectra should provide useful information about the structure and composition of fish oils. Acknowledgement We thank the Karlshamn Research Foundation for financial assistance. References I 2 3 4 5 6 7
K.F. Wollenberg (1990) J. Am. Oil Chem. Soc. 67, 487-494. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 195-199. F D. Gunstone (1991) Chem. Phys. Lipids 58, 159-167. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 201-207. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 227-229. A.G. Ferridge and J.C. Linton (1978) J. Magn. Reson. 31, 337-340. F.D. Gunstone, M.R. Pollard, C.M. Scrimgeour, N.W. Gilman and B.C. Holland, (1976) Chem. Phys. Lipids 17, 1-13.
83
High resolution NMR studies of fish oils F.D. Gunstone Chemistry Department, The University, St Andrews. F(l'e. KYI6 9ST (U.K.)
(Received April 12th, 1991; revision received May 28th, 1991; accepted May 28th, 1991) The high resolution NMR spectra of four fish oils have been recorded. Signals in the 13C-NMRspectra are assigned and attention is drawn to specific signals for the n-3 group of acids and also individually for DHA, EPA and stearidonic acid. Keywords: high resolution NMR spectra; tH; t3C; fish oils; n-3 acids; EPA: DHA; stearidonic acid.
Introduction High resolution N M R spectroscopy is used increasingly as a technique to provide insight into the nature of the mixtures present in natural oils and fats and other lipids. This applies particularly to 13C-NMR spectra. For example, Wollenberg [1] demonstrated that the chemical shifts for signals associated with the acyl carbon atoms (C 1) depend on whether the chain is ct or/3 linked to glycerol and on the nature of the unsaturation in the chain, if any. The signals for the olefinic carbon atoms also depend on whether they are present in ~ or /~ chains. Wollenberg exploited this observation by determining the distribution of saturated and unsaturated acids between oL and/~ chains. However, to get satisfactory quantitative results relaxation times had to be included in the protocol for collecting spectra and this led to data collection over 5 h or more. We came to similar conclusions from our examination of a range of synthetic glycerides alone and as simple mixtures [2]. Our spectra were obtained without relaxation periods in about 30 rain. Realising that the small changes in chemical shift resulting from unsaturation at position 9 are larger Correspondence to: F.D. Gunstone, Chemistry Department, The University, St Andrews, Fife, KYI6 9ST, U.K. Abbreviations: DHA, docosahexaenoic acid: DPA, docosapentaenoic acid; EPA (e)icosapentaenoic acid.
when the double bond is closer to the acyl group we examined seed oils containing petroselinic acid (18:1 6c) [3] and ~-linolenic acid (18:3 6c9c 12c) [41. The results were encouraging in that we could determine the amounts of these acids and their distribution between the o~ and /3 positions from our 30-min spectra. Other oils with unsaturation close to the carboxyl group include the fish oils which contain important n-3 polyene acids such as D H A (22:6 4,7,10,13,16,19), DPA (22:5 7,10,13,16,19), EPA (20:5 5,8,11,14,17), and stearidonic acid (18:4 6,9,12,15) and we now report on our spectroscopic examination of four fish oils (see Experimental). Fish oils contain complex mixtures of fatty acids and as a preliminary to this investigation we previously examined six pure n-3 polyene esters [5]. In Table I we summarise some important data (slightly revised and extended) from our earlier paper which will be used in the subsequent discussion. We will draw attention to several signals which are specific for individual acids and for groups of related acids. These may be of value in the spectroscopic examination of fish oils but improved quantitative results will require spectra collected more slowly or with the addition of relaxation agents. Our assignments are based on information taken from our previous papers and from our knowledge of the composition of the four fish oils determined by gas chromatography (Table II).
0009-3084/91/$03.50 © 1991 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
84 TABLE 1 13C-NMR data for six n-3 polyene methyl esters adapted from Ref. 5.
CI C2 C3 C4 C5 C6 C7 o~l Allylic o~2 Other Double allylic C6 o~5 Olefinic d ~03 t,,'4
22:6 (A4...)a
20:5 (A5...)a
18:4 (A6...)a
22:5 (A7...)a
20:4 (A8...)a
18:3 (A9...)a
173.46" 34.01 (22.81 )b . . 14.28
173.99 33.43* 24.80 (26.57) b . 14.29
174.03 33.97 24.60* 29.12 (26.88) b -
174.14 34.05 24.88 28.81" 29.27 (27.06) b
14.28
14.27
174.22 34.08 24.93 29.07 28.92* 29.44 (27.18) b 14.29
174.15 34.08 24.99 29.23 29.18 29.15 29.63 14.31
.
20.57 22.81(C3)
20.58 26.57(C4)
20.58 26.88(C5)
20.57 27.06(C6)
20.58 27.18(C7)
20.61 27.25(C8)
25.65 (3) c 25.59 25.56
25.64 (3) c
25.65 (2) c
25.66 (3) c
25.65 (2) c
25.67
25.56
25.56
25.56
25.56
25.57
132.02 127.03
131.99 127.05
132.02 127.06
132.00 127.04
131.88 127.17
132.00 127.02
alndicates the position of the first double bond with respect to the ester group. bThis signal is also allylic. Clndicates the number of carbon atoms represented by this signal. dUnassigned olefinic signals lying between the o~4 and oJ3 values are listed in Ref. 5. *This chemical shift is markedly different from others in the horizontal row. It is 3' to a double bond.
T A B L E I1 Major component acids for four fish oils. G C analysis on a megabore column (DB-23, J and W Scientific) using helium as carrier gas. CPL a
BT a
MAX a
CLO a
Saturated Monoene Major n-3 acids 18:4 20:5 (EPA) 22:5 (DPA) 22:6 ( D H A )
24.2 25.7
27.2 23.7
25,6 25,3
16.5 49.0
3.7 16.3 2.6 12.4
3.2 15.8 2.8 9.4
1.9 17.4 2.9 11.4
2.7 8.5 1.4 I 1.9
EPA ÷ D H A EPA + D H A
28.7 1.31
25.2 1.68
28.8 1.53
20.4 0.71
aSee Experimental.
85
Experimental The fish oils examined were: (i) CPL fish oil (provided by Lipid Teknik, Stockholm), (ii) Fish oil for Biomedical Tests provided by the National Institute of Health (U.S.A.), (iii) Max EPA, and (iv) cod liver oil (purchased locally). On the basis of gas chromatography of their methyl esters these oils contained the major component acids listed in Table II. EPA and DHA together range from 20% to 29'7,, and the ratio of EPA to DHA from 0.7 to 1.7. Cod liver oil shows the greatest difference from the remainder in its high level of monoene acids and reduced proportions of saturated and polyene acids. The spectra were obtained using CDCI3 solutions and a Brucker AM 300 spectrometer. Data were acquired over 20,000 Hz into 32K data points and zero-filled to 64K. When necessary, resolution enhancement was increased using Gaussian multiplication [6].
Results and Discussion tH-NMR spectra The fish oils show the expected range of signals in their ~H-NMR spectra but with some additional information. In addition to the usual CH3 signal at 0.85 ppm there is a triplet at 0.95 ppm arising from all the n-3 acids. Additional signals of slightly higher chemical shift also appear on the side of signals at 2.35 (C2), 2.1 (allylic), and 1.65 ppm (C3). The first of these is a signal assigned to C3 of D H A which is also an allylic carbon atom [5], the others arise from allylic or C3 hydrogens under the additional influence of a nearby double bond in the polyene acids. ~3C-NMR spectra The assignments which follow are based on our study of several vegetable oils (Gunstone, unpublished results) and on previously published information [2] including our paper on n-3 polyene esters [5]. We have abstracted some of the more important data from this paper in Table I and have there drawn attention to specific signals characteristic of each of the major polyene acids.
Glycerol, CI, C2 and C3 carbon atoms (Table III) Most vegetable oils containing only saturated, oleic, linoleic (and possibly linolenic) acids show two signals for the glycerol carbon atoms at about 68.9 (O) and 62.1 ppm (c~) with the latter twice the size of the former because it represents two carbon atoms. These are also apparent as the largest signals in the fish oils but they are accompanied by smaller signals of higher chemical shift. These probably arise from n-3 polyene acids in the B chain but we cannot be more specific. CI signals usually occur at about 173.2 (ct) and 172.8 ppm (3) with a difference of about 0.40. These are present in the fish oils and are accompanied by other signals associated with the ct chains and the B chain. On the basis of our results with methyl esters summarised in Table I we consider that the signals at 172.8 and 172.4-172.5 (difference 0.37) are due to EPA in the ct and B positions, respectively. The signal at 172.0 probably results from DHA in the B position whilst that from DHA in the c~-position (if any) would be expected around 172.4 and may overlap with EPA (3). The C2 signals for c~ and 3 chains are fairly close together (separation about 0.17 ppm) with chemical shifts about 34.2 and 34.05 ppm. We observe up to three further signals and consider that those around 33.5-33.6 and 33.3-33.4 (difference 0.18) are related to EPA in the 3 and ct position, respectively. This carbon atom is 3' to the double bond in the 5-position of EPA and is expected to be about 0.5-0.6 ppm lower than similar C2 atoms not influenced by a nearby double bond. C3 carbon atoms give two signals around 24.9 ppm (difference 0.04) for the oe and 3 chains but these two signals usually appear only after resolution enhancement. We observe three or four signals for this carbon atom. That with the highest chemical shift is the usual value corresponding to most of the acyi chains in both o~ and # positions. The remaining signals are probably due to n-3 acids. DHA will not give a signal in this region because C3 is also allylic and its chemical shift will be lower. It appears in the same range as the ¢02 carbon atoms and will be discussed further with that group of signals. Bearing in mind that EPA is the n-3 acid present in highest proportion and the
86 T A B L E I11 C h e m i c a l shifts ( p p m ) a n d intensities f o r glycerol, C I , C 2 a n d C3 c a r b o n a t o m s . CPL a Glycerol
CI
69.16 69.06
0.8 0.9
68.94 62.07
2.3 5.2
68.94 62.09
2.5 5.4
173.01
2.6
173.12
1.0 0.9
172.93 172.88 172.73 172.62
0.5
172.55 172.51 172.41 172.04
0.4 0.5 0.3 0.6
172.05
0.5
34.18 34.02 33.89 33.53 33.34
2.6 8.3 1.5 0.9 2.0
34.21 34.04 33.91 33.57 33.38
2.9 8.2 1.6 0.9 2.1
34.24 34.08 33.95 33.62 33.44
2.8 7.0 1.2 0.5 2.2
24.90 24.78
9.1 1.8
24.89 24.78
8.7 1.9
24.92
24.72 24.49
2.9 1.2
24.72 24.49
2.5 1.4
24.76 24.52
0.4
69.23
CLO a
1.2 0.9
172.43 171.96
C3
MAX a
69.16 69.06
172.80 172.63 -
C2
BT a
0.8
-
Ass. b
69.23
1.4
-
69.01 62. I 1
3.4 7.4
/3 t~
4.6
ct
69.00 62.14
2.4 4.9
3.5
173.14
2.5
173.02
0.5 1.1 1.5 0.2
172.89 172.75 -
0.9 1.3
172.64 -
1.8
171.97
0.9
34.21 34.05
3.7 10.8
33.58 33.40
0.9 1.3
7.5
24.91 24.82
12.3 2.4
2.8 1.0
24.75 24.52
2.0 0.9
-
-
E P A c~ /~ EPA D H A /~ /~ ct EPA/3 E P A ct /~ c~ D P A etc. EPA 18:4
aSee E x p e r i m e n t a l . b S o m e o f these a s s i g n m e n t s a r e o n l y t e n t a t i v e (see text),
relative values of the chemical shifts for the methyl esters (Table I) we have assigned the signals as follows: 24.5, 18:4 (stearidonic acid); 24.75, EPA; 24.8, DPA and possibly 20:4 and 22:4.
o~1, o~2 and oJ3 signals (Table IV) The two 601 signals correspond to n-3 (14.3 ppm) and n-9/saturated (14.2 ppm). These oils contain only low levels of n-6 polyene acids and since the signal for this group is not well separated from that for the n-9/saturated chain (difference about 0.04) it is not apparent in these spectra. The major o~2 signal is derived from n-9/saturated acids. One oil shows a signal for n-6 acids. The o~2 atom in the important n-3 acids is also allylic and is discussed along with that group of
signals. The signal at 22.8 ppm observed with one oil may be due to DHA C3 which is influenced by its position with respect to the ester group and the adjacent double bond. The o~3 atom for n-3 acids is olefinic (see appropriate discussion). A small signal for n-6 acids is apparent in three of the spectra. The major signal expected at 31.9-32.0 (n-9 and saturated acids) is accompanied by a smaller but significant signal about 31.85 ppm but we have no explanation of this at the present time.
Allylic signals (Table IV) Allylic signals are easily divided into two categories depending on whether the carbon atoms are adjacent to one or two double bonds. These
87
TABLE Chemical
IV s h i f t s ( p p m ) a n d i n t e n s i f i e s f o r o~1, oJ2, oJ3 a n d a l l y l i c c a r b o n
CPL a
¢01
w 2
CLO a
Ass. b
14.30
5.5
14.29
4.3
14.26
3.5
14.28
4.6
10.2
14.13
9.1
14.10
7.9
14.11
13.7
-
13.81
0.6
-
-
-
22.92
0.2
-
-
-
22.81
1.0
-
-
22.72
9.8
22.72
22.62 32.83
1.0 0.2
-
10.6
-
Allylic
MAX a
14.14
22.74 w3
BT a
atoms.
9.2
32.19
0.2
-
9.4 2.7
31.97 31.83
7.5 3.3
31.97 31.84
7.2 2.1
31.57
0.9
31.56
0.9
31.57
0.7
30.02 -
0.4
. 27.47
0.2
27.41
0.4
27.21
8.7 6.2
27.25 27.21
7.4 5.6
27.27 27.22
27.07
0.6
27.06
0.7
26.95
0.4
26.95
26.88
1.5
26.53
3.1
27.25
DHA 15.6
31.97 31.85
11.8 1 3.7 J
n-9, s a t
-
n-6
-
-
-
-
-
.
.
C3
n-9, s a t n°6 -
.
6.7 4.5
-
27.27 -
17.21
-
27.05
1.0
AIlylic
0.3
-
-
26.87
1.6
26.89
1.2
26.89
1.2
18:4 ( C 5 )
26.53
2.7
26.57
2.6
26.56
2.0
EPA (C4)
-
J DPA (C6)
25.94
0.3
25.67
21.2
25.67
18.3
25.70
14.6
25.70
17.3
25.58
7.5
25.58
6.1
25.61
4.9
25.60
6.6
-
n-9, s a t
-
31.99 31.85
-
22.73
n-3
-
]Double B Lallylic ['DHA
[,o5
C 6 n-3
B
-
25.38
0.3
-
-
-
25.13
0.2
-
-
-
-
25.10
0.2
-
-
-
20.59
4.0
20.60
20.59
4.8
aSee Experimental. bSome of these assignments
3.1
20.60
-
4.0
n-3 w2
a r e o n l y t e n t a t i v e ( s e e text).
signals usually occur at about 27.2-27.3 (sometimes appearing as two signals) and 25.6-25.7 ppm. In these fish oils the significant signals at about 27.25, 27.20 and 25.70 ppm correspond to these major allylic signals. In addition the signal of 20.6 ppm derives from the w2 carbon atom in all n-3 acids. There remain several signals for which we make the following suggestions: 25.6 ppm, DHA C6 and w5 carbon atoms in n-3 acids; 26.5-26.6 ppm, EPA C4; 26.9 ppm, stearidonic acid C5; 26.95-27.1 ppm, DPA C6 and other n-3 acids. The allylic carbon atoms at C6 (DHA), C4
(EPA), and C5 (stearidonic acid) come under the additional influence of the ester group and have lower chemical shifts than when this influence is absent. As already indicated the C3 (DHA) signal overlaps with the w2 signals.
Olefinic signals (Table V) Each of the fish oils gives 15-20 olefinic signals (before resolution enhancement) and this has proved to be the most difficult part of the spectrum to assign [5]. For the sake of completeness we present all our results in Table V but we are able to make
88 TABLE V Chemical shifts (ppm) and intensities for olefinic carbon atoms. CPL a 131.92
BT a 4.0
-
130.06 129.95 129.88 129.78 129.67 129.48 129.45 128.99 128.77 128.53 128.25 128.14 128.06 127.98 127.86 127.67 127.59 127.06
0.6 3.7 2.3 1.8 3.4 2.0 2.1 3.4 3.4 5.0 8.5 5.7 7.6 3.9 5.9 2.5 0.6 4.8
131.97 130,42 130.27 130,11 129.98 129.91 129.81 129.69 129.51 129.47 129.10 129.00 128.78 128.56 128.41 128.27 128.16 128.08 127.99 127.88 127.67 . 127.06
MAX a 3.9 0.3 0.2 0.8 4.2 1.6 1.3 3.4 1.5 1.7 0.2 3.0 3.0 4.4 1.3 7.0 4.8 6.1 2.6 4.6 1.6 . 4,1
132.02 130.02 129.94 129.84 129.72 129.51 129.03 128.81 128.61 128.32 128.24 128.20 128.12 128.02 127.92 127.70 . 127.10
CLO a 3.3
131.96 129.99 129.91 129.80 129.70 129.49 129.03 128.79 128.57 128.29 128.17 128.10 128.00 127.90 127.70
3.3 1.7 1.4 2.9 1.5
2.6 2.7 3.7 6.2 3.4 4.1 5.6 2.3 4.0 1.6
Ass.b 4.2
6.2 5.3] 4.9 J 5.9 2.5
2.3 2.3 4.5 7.7 4.0 7.0 3.6 4.7 2.6
n-3 ¢~3 OI0 Monoene 09 -
. 3.3
127.09 4.2
n-3
aSee Experimental. bSome of these assignments are only tentative (see text). O = oleic (and other A9 acids).
only a limited number of assignments. The signals of highest (131.9-132.0 ppm) and lowest (127.05-127.1 ppm) chemical shift are associated w i t h t h e oJ3 a n d oJ4 c a r b o n a t o m s r e s p e c t i v e l y in all t h e n-3 p o l y e n e a c i d s . S i g n a l s a t 1 2 9 . 9 5 - 1 3 0 . 0 p p m and 129.65-129.7 ppm arise from CI0 and C9 r e s p e c t i v e l y in o l e i c a c i d a n d o t h e r A 9 m o n o e n e s . W e f u r t h e r b e l i e v e t h a t t h e s i g n a l s a t 129.9 a n d 129.8 p p m c o m e f r o m o t h e r m o n o e n e a c i d s w i t h unsaturation further from the carboxyl group than A9. T h e h i g h e r i n t e n s i t i e s o f t h e s e f o u r m o n o e n e s i g n a l s i n c o d l i v e r oil is c o n s i s t e n t w i t h t h e h i g h level o f m o n o e n e a c i d s i n t h a t oil ( T a b l e 1I).
General comments on assignments In Table VI we compare some results for methyl e s t e r s w i t h t h o s e f o r g l y c e r o l e s t e r s in r e s p e c t o f c a r b o n a t o m s 2 a n d 3. T h e d i f f e r e n c e s in c h e m i c a l s h i f t s f o r t h e s e t w o t y p e s o f e s t e r s a r e s m a l l a n d we feel j u s t i f i e d in a p p l y i n g d a t a a v a i l a b l e f o r m e t h y l esters for assignments of glycerol ester spectra. A l s o , in a n y g r o u p o f s i g n a l s f o r a c a r b o n a t o m we compare the major chemical shift (for saturated a n d A9 o r n - 9 acyl c h a i n s ) w i t h t h a t o f s m a l l e r signals in the same region. A reduction of -0.4-0.5 p p m i n d i c a t e s t h a t t h e c a r b o n a t o m is 7 t o d o u b l e b o n d . T h i s l o w e r v a l u e is a p p a r e n t , f o r
89 TABLE VI Chemical shifts (ppm) for C2 and C3 atoms in methyl and glycerol esters. C2 Methyl ester Saturated C13-C21 a CI2, CI4, CI6 b Oleic b Linoleic b 18:1 (6) b'c 18:3 (6,9,12) b'd
34.18 34.1 ! 34.10 34.01 33.97
C3 Giyceride
34.24, 34.20, 34.19, 34.11, 34.06,
34.67 34.04 34.01 33.95 33.91
Methyl ester
25.11 24.98 24.94 24.63 24.63
Glyceride
24.92 24.87 24.85 24.53 24.50
aRef. 7. bUnpublished information (F.D.G.). CRef. 3. dRef. 4.
example, at C1, for DHA ( A 4 . . . ) , C2 for EPA (A5...), and C3 for stearidonic acid (A6...). Conclusion To exploit the 13C-NMR spectra of fish oils we need (a) to assign all or most of the chemical shifts with a view to identifying those which are specific for one fatty acid or for a group of acids and (b) to improve the quantitative aspects of the spectra. This paper is devoted to the assignments: most of these are quite firm, but some are only tentative, and some have yet to be made. Finally we list those signals which provide spectroscopic information about the n-3 acids as a group or about individual members of that group. n-3 acids: DHA: EPA: Stearidonic:
~01 and olefinic signals CI(#) and C3 (in the oJ2 group) CI (a and #), C2 (c~ and ~/), C3, and ailylic (C4) C3 and allylic (C5)
It is clear that the t3C-NMR spectra of fish oils
contain useful information relating to the important n-3 acids, both as a group and individually. With improved quantitation 13C-NMR spectra should provide useful information about the structure and composition of fish oils. Acknowledgement We thank the Karlshamn Research Foundation for financial assistance. References I 2 3 4 5 6 7
K.F. Wollenberg (1990) J. Am. Oil Chem. Soc. 67, 487-494. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 195-199. F D. Gunstone (1991) Chem. Phys. Lipids 58, 159-167. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 201-207. F.D. Gunstone (1990) Chem. Phys. Lipids 56, 227-229. A.G. Ferridge and J.C. Linton (1978) J. Magn. Reson. 31, 337-340. F.D. Gunstone, M.R. Pollard, C.M. Scrimgeour, N.W. Gilman and B.C. Holland, (1976) Chem. Phys. Lipids 17, 1-13.