Comparison of Methylation Methods for the Quantitation of Conjugated Linoleic Acid Isomers

1993 ◽  
Vol 76 (3) ◽  
pp. 644-649 ◽  
Author(s):  
Nalur Chandrasekaran Shantha ◽  
Eric Andrew Decker ◽  
Bernhard Hennig
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Sodium Methoxide ◽  
Boron Trifluoride ◽  
Acetyl Chloride ◽  
Octadecadienoic Acid ◽  
Methyl Esters ◽  
Low Concentrations ◽  
Cla Isomers ◽  
In Cis

Abstract Four methylation methods were evaluated for use in the gas chromatographic (GC) quantitation of conjugated linoleic acid (CLA) isomers, which are potential anticarcinogen. The methods were (1) sodium methoxide in methanol (NaOMe-MeOH), (2) American Oil Chemists' Society (AOCS) procedure Ce 2-66, which involves methanolic sodium hydroxide followed by boron trifluoride in methanol, (3) tetramethylguanidine in methanol (TMG-MeOH), and (4) direct transesterification with methanolbenzene- acetyl chloride (DAC). Purified methyl esters of isomerized linoleic acid containing 86% CLA isomers were methylated and analyzed by GC. The AOCS and DAC methods resulted in 3 and 50% losses in cis-9,trans-11-octadecadienoic acid (9c, 111 CLA isomer) and trans-10,cis-12 octadecadienoic acid (10t, 12c CLA isomers), respectively. Compared with the control, the AOCS and DAC methods increased the yield of the trans,trans CLA isomers (trans-9,trans-11- and trans-10, trans-12-octadecadienoic acid) by 1.07-fold and a 10-fold, respectively. A non-CLA artifact that eluted close to CLA peaks was formed during methylation by the AOCS and DAC methods. Thus, the DAC and AOCS methods are not suitable for quantitation of CLA isomers. The NaOMe-MeOH and TMG-MeOH methods, however, are suitable for quantitation of CLA isomers in fats containing low concentrations of free fatty acids.

scholarly journals Improved Identification of Conjugated Linoleic Acid Isomers Using Silver-Ion HPLC Separations

2004 ◽  
Vol 87 (2) ◽  
pp. 563-568 ◽  
Author(s):  
Pierluigi Delmonte ◽  
Martin P Yurawecz ◽  
Magdi M Mossoba ◽  
Cristina Cruz-Hernandez ◽  
John K G Kramer
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Mobile Phase ◽  
High Performance ◽  
Methyl Esters ◽  
Solvent Mixture ◽  
Silver Ion ◽  
Dead Volume ◽  
Relative Retention ◽  
Cla Isomers

Abstract Silver-ion high-performance liquid chromatography (Ag+-HPLC) has been shown to be effective in the resolution of most of the isomers of conjugated octadecadienoic acids (18:2), also known as conjugated linoleic acid (CLA). The CLA isomers identified in natural fats from ruminants are a mixture of numerous positional and geometric isomers from 7,9- to 12,14–18:2. Ag+-HPLC separates both geometric (trans,trans < cis/trans < cis,cis) and positional CLA isomers using the mobile phase hexane/acetonitrile (99.9:0.1). The elution volumes for the CLA isomers were not only affected by the concentration of acetonitrile (in the prepared mobile phase) but also with successive runs during the day using a prepared mobile phase batch, due to the partial solubility of acetonitrile in hexane. However, this drift does not affect the relative resolution of the CLA isomers. The addition of diethyl ether to the mobile phase partly stabilizes the solvent mixture. In order to facilitate the interpretation of Ag-+HPLC chromatograms, the relative retention volumes (RRV) were calculated for each CLA isomer. Toluene was added to all the test portions and served as an estimator of dead volume, whereas the elution of the ubiquitous 9c,11t-CLA isomer was chosen as unity (1.00). Expressing the elution of all the CLA isomers as their RRV greatly helped to standardize each CLA isomer, resulting in relatively small coefficients of variation (% CV) for the trans,trans (<1.5%) and cis/trans (<0.5%) CLA isomers. The identification of the CLA isomers was further facilitated by synthesis of authentic CLA isomers. All the geometric CLA fatty acid methyl esters (FAME) from positions 6,8- to 13,15-CLA were commercially available or synthesized by a combination of partial hydrazine reduction of known polyunsaturated fatty acids followed by alkali isomerization, isolation of products, and further iodine-catalyzed geometric isomerization. Based on expressing the elution volume as RRV and the availability of the synthetic CLA isomers, a unique reversal of the elution order of the c/t CLA isomers was found. It is also proposed that the retention times of CLA isomers by gas chromatography (GC) should be expressed as their relative retention times (RRT) relative to methyl γ-linoleneate. The availability of CLA reference materials and the application of RRV and RRT to Ag+-HPLC and GC separations, respectively, will greatly improve in the identifications of CLA isomers.

scholarly journals Conjugated Linoleic Acid Accumulation via 10-Hydroxy-12-Octadecaenoic Acid during Microaerobic Transformation of Linoleic Acid by Lactobacillus acidophilus

2001 ◽  
Vol 67 (3) ◽  
pp. 1246-1252 ◽  
Author(s):  
Jun Ogawa ◽  
Kenji Matsumura ◽  
Shigenobu Kishino ◽  
Yoriko Omura ◽  
Sakayu Shimizu
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Lactobacillus Acidophilus ◽  
Octadecadienoic Acid ◽  
Total Fatty Acids ◽  
Cla Isomers ◽  
Microaerobic Conditions ◽  
Almost All

ABSTRACT Specific isomers of conjugated linoleic acid (CLA), a fatty acid with potentially beneficial physiological and anticarcinogenic effects, were efficiently produced from linoleic acid by washed cells ofLactobacillus acidophilus AKU 1137 under microaerobic conditions, and the metabolic pathway of CLA production from linoleic acid is explained for the first time. The CLA isomers produced were identified as cis-9, trans-11- ortrans-9, cis-11-octadecadienoic acid andtrans-9, trans-11-octadecadienoic acid. Preceding the production of CLA, hydroxy fatty acids identified as 10-hydroxy-cis-12-octadecaenoic acid and 10-hydroxy-trans-12-octadecaenoic acid had accumulated. The isolated 10-hydroxy-cis-12-octadecaenoic acid was transformed into CLA during incubation with washed cells of L. acidophilus, suggesting that this hydroxy fatty acid is one of the intermediates of CLA production from linoleic acid. The washed cells of L. acidophilus producing high levels of CLA were obtained by cultivation in a medium containing linoleic acid, indicating that the enzyme system for CLA production is induced by linoleic acid. After 4 days of reaction with these washed cells, more than 95% of the added linoleic acid (5 mg/ml) was transformed into CLA, and the CLA content in total fatty acids recovered exceeded 80% (wt/wt). Almost all of the CLA produced was in the cells or was associated with the cells as free fatty acid.

Conjugated linoleic acid in ewe milk fat

2005 ◽  
Vol 72 (4) ◽  
pp. 415-424 ◽  
Author(s):  
Pilar Luna ◽  
Javier Fontecha ◽  
Manuela Juárez ◽  
Miguel Angel de la Fuente
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Milk Fat ◽  
High Performance ◽  
Methyl Esters ◽  
Complex Mixture ◽  
Gas Chromatography Mass Spectrometry ◽  
Mass Spectral ◽  
Total Fatty Acids ◽  
Cla Isomers

Ewe milk fat from five different herds was studied to determine the content of conjugated linoleic acid (CLA) isomers. Research was carried out by combining gas chromatography-mass spectrometry (GC-MS) of fatty acid methyl esters (FAME) and 4,4-dimethyloxazolyne derivatives (DMOX) with silver ion-high performance liquid chromatography (Ag+-HPLC). Reconstructed mass spectral profiles of CLA characteristic ions from DMOX were used to identify positional isomers and Ag+-HPLC to quantify them. Total CLA content varied from 0·57 to 0·97 g/100 g of total fatty acids. FAME and DMOX were separated into a complex mixture of minor isomers and major rumenic acid (9-cis 11-trans C18:2) by GC-MS using a 100-m polar capillary column. Rumenic acid would represent more than 75% of total CLA. 11-trans 13-trans, 11–13 cis/trans plus trans/cis and 7–9 cis/trans plus trans/cis were the main CLA isomers after rumenic acid. Minor amounts of 8–10 and 10–12 C18:2 isomers were also found. Although most of the isomers were present in each herd's milk, differences in content were observed for some CLA species.

Conjugated linoleic acid (CLA) isomers in human adipose tissue

1997 ◽  
Vol 205 (6) ◽  
pp. 415-418 ◽  
Author(s):  
J. Fritsche ◽  
Magdi M. Mossoba ◽  
Martin P. Yurawecz ◽  
John A. G. Roach ◽  
Najibullah Sehat ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Human Adipose Tissue ◽  
Cla Isomers

A new conjugated linoleic acid isomer, 7 trans, 9 cis-octadecadienoic acid, in cow milk, cheese, beef and human milk and adipose tissue

Lipids ◽  
1998 ◽  
Vol 33 (8) ◽  
pp. 803-809 ◽  
Author(s):  
Martin P. Yurawecz ◽  
John A. G. Roach ◽  
Najibullah Sehat ◽  
Magdi M. Mossoba ◽  
John K. G. Kramer ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Linoleic Acid ◽  
Human Milk ◽  
Conjugated Linoleic Acid ◽  
Octadecadienoic Acid ◽  
Cow Milk ◽  
Conjugated Linoleic Acid Isomer ◽  
Linoleic Acid Isomer

scholarly journals Isomerisation of cis-9 trans-11 conjugated linoleic acid (CLA) to trans-9 trans-11 CLA during acidic methylation can be avoided by a rapid base catalysed methylation of milk fat

2008 ◽  
Vol 75 (3) ◽  
pp. 354-356 ◽  
Author(s):  
Michael R F Lee ◽  
John K S Tweed
Keyword(s):  
Fatty Acid ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Milk Fat ◽  
Fatty Acid Methyl Esters ◽  
Methyl Esters ◽  
The Other ◽  
Gas Liquid Chromatography ◽  
Freeze Dried ◽  
Acidic Conditions

This study investigated the evolution of trans-9 trans-11 conjugated linoleic acid (CLA) from cis-9 trans-11 CLA during methylation and its avoidance through a rapid base methylation of milk fat. The study examined three conditions shown to result in loss of cis-9 trans-11 CLA during methylation namely: temperature, methylation time, water contamination in old reagents and acidic conditions. Three techniques currently used for the conversion of milk fat into fatty acid methyl esters for analysis of CLA content by gas liquid chromatography and a fourth procedure designed to eliminate acidic conditions and to limit methylation temperature and time were used. The four methods were: (i) acidic methylation (AM); (ii) acidic and basic bimethylation with fresh reagents (FBM); (iii) acidic and basic bimethylation with pre-prepared reagents (PBM) and (iv) basic methylation (BM). Each regime was carried out on six milk samples over two periods and methylated 1 ml freeze-dried milk (n=12 per regime). Total CLA was not different across methylation regimes (0·30 mg/ml). Isomer cis-9 trans-11 was higher (P<0·01) with BM than the other regimes and lowest with AM: 21·2, 17·8, 18·8 and 14·7 mg/100 ml for BM, FBM, PBM and AM, respectively. The inverse relationship was shown for trans-9 trans-11 with higher (P<0·001) amounts with AM than the other regimes and lowest with BM: 0·57, 2·55, 2·36 and 3·69 mg/100 ml for BM, FBM, PBM and AM, respectively. The trans-10 cis-12 isomer was also shown to alter with methylation procedure being higher (P<0·001) with AM than the other regimes: 0·43, 0·47, 0·29 and 1·20 mg/100 ml for BM, FBM, PBM and AM, respectively. Validation with known CLA free fatty acid and triacylglycerol standards confirmed that AM resulted in conversion of cis-9 trans-11 to trans-9 trans-11, and also elevated trans-10 cis-12 whilst BM of triacylglycerol CLA did not isomerise cis-9 trans-11 and was comparable to FBM.

scholarly journals In silico characterization of linoleic acid biotransformation to rumenic acid in food derived Lactobacillus plantarum YW11

2020 ◽  
Author(s):  
Tariq Aziz ◽  
Abid Sarwar ◽  
Muhammad Fahim ◽  
Sam Al Dalali ◽  
Zia Ud Din ◽  
...  
Keyword(s):  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Lactobacillus Plantarum ◽  
In Silico ◽  
Octadecadienoic Acid ◽  
Scale Up ◽  
Octadecenoic Acid ◽  
Acid Ethyl Ester ◽  
Dependent Manner ◽  
In Silico Characterization

Lactobacillus plantarum YW11 capability to convert linoleic acid into conjugated linoleic acid and other metabolites was studied in a dose-dependent manner by supplementing LA at different concentrations. L. plantarum YW11 displayed a uniform distinctive growth curve of CLA and other metabolites at concentrations of LA ranging from 1% (w/v) to 10% (w/v), with slightly increased growth at higher LA concentrations. The biotransformation capability of L. plantarum YW11 evaluated by GC-MS revealed a total of one CLA isomer, i.e. 9-cis,11-trans-octadecadienoic acid, also known as the rumenic acid (RA), one linoleic acid isomer (linoelaidic acid), and LA metabolites: (E)-9-octadecenoic acid ethyl ester, trans, trans-9,12-octadecadienoic acid, propyl ester and stearic acid. All the metabolites of linoleic acid were produced from 1 to 10% LA supplemented MRS media, while surprisingly the only conjugated linoleic acid compound was produced at 10% LA. To assess the presence of putative enzymes, responsible for conversion of LA into CLA, in silico characterization was carried out. The in silico characterization revealed presence of four enzymes (10-linoleic acid hydratase, linoleate isomerase, acetoacetate decarboxylase and dehydrogenase) that may be involved in the production of CLA (rumenic acid) and LA isomers. The biotransformation ability of L. plantarum YW11 to convert LA into RA has great prospects for biotechnological and industrial implications that could be exploited in the future scale-up experiments.

scholarly journals  A new internal standard for HPLC assay of conjugated linoleic acid in animal tissues and milk

2011 ◽  
Vol 56 (No. 1) ◽  
pp. 23-29 ◽  
Author(s):  
M. Czauderna ◽  
J. Kowalczyk ◽  
M. Marounek ◽  
J.P. Michalski ◽  
A.J. Rozbicka-Wieczorek
Keyword(s):  
Fatty Acids ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Biological Samples ◽  
Mobile Phase ◽  
Sorbic Acid ◽  
Internal Standard ◽  
Silver Ion ◽  
Animal Origin ◽  
Cla Isomers

A new method for the quantification of underivatized conjugated linoleic acid (CLA) isomers and CLA-metabolites by silver ion liquid chromatography (Ag<sup>+</sup>-HPLC) with photodiode array detection (DAD) is described. Conjugated fatty acids (CFA) and sorbic acid as the internal standard (IS) were separated on two 5 &mu;m Chrompac ChromSpher Lipids columns (250 &times; 4.6 mm). Biological samples were hydrolyzed with 1M KOH in methanol and 2M KOH in water at room temperature for 12 h. Hydrolyzates were acidified and the free fatty acids were extracted with dichloromethane. The organic solvent was removed and then the residue was re-dissolved in hexane and centrifuged. The supernatant was injected onto the columns. The mobile phase of 1.6% acetic acid and 0.0125% acetonitrile in hexane was chosen as the optimum mobile phase for fractionation of IS, CLA isomers and CLA-metabolites in all assayed biological samples. The use of two silver ion-exchange columns with direct UV detection (Ag<sup>+</sup>-HPLC-DAD) offers satisfactory precision of the IS quantification and low limits of detection of IS and CLA isomers (0.60 and 0.21&ndash;0.35 ng, respectively). The presented simple Ag<sup>+</sup>-HPLC-DAD method with sorbic acid as the IS can be used for direct determination of underivatized CLA isomers in specimens of animal origin. &nbsp;

Dietary effect on bovine milk fat conjugated linoleic acid content

2000 ◽  
Vol 25 ◽  
pp. 283-293
Author(s):  
F. Lawless ◽  
J.J. Murphy ◽  
S. Fitzgerald ◽  
B. O’Brien ◽  
R. Devery ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Linoleic Acid ◽  
Conjugated Linoleic Acid ◽  
Growth Rates ◽  
Milk Fat ◽  
Methyl Esters ◽  
Bovine Milk ◽  
Fold Increase ◽  
Linoleic Acid Content ◽  
Grass Growth

AbstractConjugated linoleic acid (cis-9, trans-11-C18:2; CLA) in milk arises as a result of microbial biohydrogenation of dietary linoleic and linolenic acids in the rumen (Kepler and Tove, 1967). Milk fat CLA concentrations were significantly (P<0.05) higher when cows were fed silage supplemented with pulp’n brew (a mixture of brewers grains, a by-product of the brewing industry, and sugar beet pulp in dry matter proportions of 0.65:0.35), compared with silage alone (Trial 1). Intake of spring grass resulted in a 2.1–fold increase in milk fat CLA concentrations over cows receiving autumn grass. Throughout lactation in Trial 2, spring calving cows produced higher milk fat CLA concentrations (from 0.5-2.7 g/100 g fatty acid methyl esters (FAME)) than autumn calving cows (0.3-1.7 g/100 g FAME); the former having spent 80% and the latter 50% of lactation on pasture. The CLA content was higher in late lactation milk compared with early lactation milk in both herds. There were no significant differences in milk yields or milk constituent yields between the herds. Manufacturing milk obtained between March and September was analyzed for milk fatty acid composition and the data correlated with grass growth throughout the season. Significant positive correlations were obtained between grass growth rates and concentrations of CLA and linolenic acid in milk fat. The data indicate that seasonal variation in milk fat CLA concentrations may be attributed to variation in pasture growth rates.

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