| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Estação Zootécnica Nacional, Instituto Nacional de Investigação Agrária e das Pescas, 2005-048 Vale de Santarém, Portugal
1 Corresponding author: rjbbessa{at}mail.telepac.pt
 |
ABSTRACT |
|---|
 TOP ABSTRACT ACKNOWLEDGEMENTSREFERENCES |
|---|
Key Words: gas chromatography-tandem mass spectrometry • heptadecenoic acid • milk fat • muscle
Heptadecenoic acid (17:1) has long been recognized as minor constituent of ruminant fats (Shorland and Jessop, 1955). Recently it was proposed, along with other odd-chain fatty acids, as a potential marker of microbial biomass (Vlaeminck et al., 2005). The odd-chain fatty acids are assumed to be mainly of rumen microbial origin and, after intestinal absorption, they are deposited in tissues or exported to milk fat. However, the origin of 17:1 is not clear. Recently Fievez et al. (2003) suggested that it could be an endogenous product of 
9-desaturation of heptadecanoic acid (17:0). This would imply that the main 17:1 isomer in ruminant fats is 17:1 cis-9. However, in spite of the earliest reports indicating that 17:1 cis-9 is the main isomer in ruminant fat (Hansen et al., 1960; Hay and Morrison, 1970, 1973), most of the recent reports are ambiguous. In fact, most reports either do not present 17:1 or leave it undefined, whereas others call it 17:1 cis-10 (Franklin et al., 1999; Marks et al., 2004; Loor et al., 2005). The fact that the methyl ester standard available from commercial chemical companies is the cis-10 isomer, which probably coelutes with cis-9 isomer in some gas chromatography (GC) systems certainly contributes to the ambiguity. Our objective was to clarify which 17:1 isomers are predominant in ruminant fats through GC chemical ionization-tandem mass spectrometry techniques and to determine if common GC conditions can resolve 17:1 isomers.
Samples tested were chosen from several experimental sets analyzed for fatty acid composition in our laboratory, and included milk fat from bovine, ovine, and caprine, and intramuscular fat of lambs, kids, and beef. Lipid extraction in samples was conducted according to Folch et al. (1957) and methyl esters were prepared using a base-catalyzed transesterification procedure described by Christie (1993). The cis isomers of ovine milk fat were separated by preparative TLC on plates impregnated with 20% AgNO3 and were located by spraying with 2',7'-dichlorofluorescein solution in isopropanol. Samples were also spiked with 17:1 cis-10 standard obtained from Sigma Chemical Co. (St. Louis, MO). Fatty acid methyl esters were resolved in 3 GC systems: 1) Varian Saturn 2000 (Varian Inc., Walnut Creek, CA) equipped with ion-trap mass detector and a BPX-70 (SGE Chromatography Supplies, Austin, TX) capillary column (60 m, 0.25 mm i.d., 0.25-µm film thickness); 2) Agilent HP6890 (Agilent Tech. Inc., Palo Alto, CA) equipped with a flame-ionization detector and a CP-Sil 88 (Chrompack CP 7489, Varian Inc.) capillary column (100 m, 0.25 mm i.d., 0.20-µm film thickness); and 3) Varian 3800 GC equipped with a flame-ionization detector and an OmegaWax 250 (Supelco, Bellefont, CA) capillary column (30 m, 0.25 mm i.d., 0.25-µm film thickness). Helium was the carrier gas in all systems.
Structural analysis of 17:1 methyl ester isomers was conducted in the chemical ionization-tandem mass spectrometry mode of the Varian Saturn 2000 system. Ion-trap parameters used in all analyses presented included: trap temperature, 170°C; manifold temperature, 80°C; transfer line temperature, 200°C; and axial modulation amplitude, 3.8 V. Acetonitrile was used as chemical ionization (CI) reagent gas with the following CI parameters: CI storage level, 19 m/z; ejection amplitude, 15 m/z; maximum ionization time, 2,000 µsec; maximum reaction time, 40 ms; prescan ionization time, 200 µsec; and target total ion current, 5,000 counts. Additional tandem mass spectrometry parameters were as follows: emission current, 40 µA; scan time, 0.34 s; mass isolation window, 3 m/z; and excitation storage level, 85 m/z. The resonant excitation amplitudes used to collisionally dissociate the (M + 54)+ ions varied from 1.60 to 2.0 V.
Samples of ovine, bovine, and caprine origin were resolved in the 3 GC systems with and without addition of 17:1 cis-10 methyl ester standard (Figure 1
). The presence of cis-10 isomer was not detected in all samples studied. All samples spiked with 17:1 cis-10 showed a well-defined peak (presumably 17:1 cis-9) and occasionally other minor peaks (presumably 17:1 cis-8) preceding the 17:1 cis-10 peak. Thin-layer chromatography isolation of the cis fraction of ovine milk fat samples allowed the evidence of the cis-8 isomer peak. Precht and Molkentin (2000), using a 100-m CP-Sil 88 column, reported good resolution between cis-8 and cis-9 17:1 isomers in human milk fat. Hay and Morrison (1970) reported that cis-8 was the second major 17:1 isomer in cow milk fat, comprising 20.1% of total heptadecenoates. We found in the cis fraction of ovine milk that the cis-8 isomer peak area was about 15% of the cis-9 area. As shown in Figure 1, the 100-m CP-Sil 88 and the 30-m OmegaWax columns unequivocally resolved cis-10 and cis-9 isomers as well as a minor amount of cis-8. The chromatographic resolution was less complete when the 60-m BPX-70 column was used.
|
). Collision dissociation of the [M+54]+ ion by CI-tandem mass spectrometry yields 2 diagnostic fragments (Figure 3) allowing unambiguous localization of the double bond. Figure 3 presents CI-tandem mass spectrometry spectra of standard 17:1 cis-10 (panel A), 17:1 cis-9 (panel B) and cis-8 (panel C) of ovine milk fat samples. The diagnostic ions for the cis-9 isomer appear at m/z 252 and 194, respectively, and analogous ions for the cis-10 standard isomer occur at m/z 266 and 180, respectively. The resolution of cis-8 isomer was not perfect with the 60-m BPX 70 column (Figure 1
), but it was possible to identify the expected diagnostic ions (m/z 238 and 208).
|
|
|
ACKNOWLEDGEMENTS |
|---|
|
TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
|---|
Received for publication July 1, 2005. Accepted for publication August 30, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTACKNOWLEDGEMENTSREFERENCES |
|---|
-9 heptadecenoic acid from lamb caul fat. Nature 176:737.[Medline]This article has been cited by other articles:
|
|
 |
|
  O. A. Rego, S. M. M. Regalo, H. J. D. Rosa, S. P. Alves, A. E. S. Borba, R. J. B. Bessa, A. R. J. Cabrita, and A. J. M. Fonseca Effects of Grass Silage and Soybean Meal Supplementation on Milk Production and Milk Fatty Acid Profiles of Grazing Dairy Cows J Dairy Sci, July 1, 2008; 91(7): 2736 - 2743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. J. Cabrita, R. J. B. Bessa, S. P. Alves, R. J. Dewhurst, and A. J. M. Fonseca Effects of Dietary Protein and Starch on Intake, Milk Production, and Milk Fatty Acid Profiles of Dairy Cows Fed Corn Silage-Based Diets J Dairy Sci, March 1, 2007; 90(3): 1429 - 1439. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
