A simplified technique for analysis by alkali isomerization

Summary Potassium tertiary butoxide (1.1 M, 15.5%) in t-butanol readily isomerized linoleic and linolenic acids at 60°C. The absorptivity value for linoleic acid during isomerization rose rapidly, then slowly reached a maximum (88.5) after three days. Maximum absorptivity values for linolenic acid at both 233 mμ (68.5) and at 268 mμ (65.0) were attained at 12 hrs.; thereafter absorptivity at both wavelengths decreased with time. Glycerol also reacted with potassium-t-butoxide under the experimental conditions to produce an absorption maximum at 267 mμ; however this absorbance was easily destroyed by treatment with hydrochloric acid. A simple procedure has been described for isomerization with the alkoxide in a reagent bottle at 60° for 20 hrs. Analyses for linoleic and linolenic acids in five seed oils isomerized by the “bottle method” agreed well with results obtained by the A.O.C.S. KOH-glycol method. Journal Paper No. 1346 of the Purdue Agricultural Experiment Station, Lafayette, Ind.     

Further studies on the isomerization of polyunsaturated fatty acids by potassium tertiary butoxide

Summary A temperature and time study revealed that the best conditions for the isomerization and estimation of polyunsaturated acids are heating in a sealed tube for 2 hrs. at 140° with potassium-t-butoxide in t-butanol. Under these conditions conjugation of linolenic and arachidonic acids appears to have attained completion. With linoliec acid a higher degree of conjugation than by other methods and a shift of peak to lower wavelength (231–232 mμ) are observed. Extinction coefficients obtained by this method for isomerized pure linoleic (k₂₃₂, 98.0) and linolenic (k₂₆₈, 138.4) acids are the highest so far reported. Ultraviolet and infrared examination of the conjugated trienes isolated from isomerized linolenic acid concentrate showed that, under these conditions, trienes similar to both alpha- and beta-eleostearic acids are produced. Analyses of samples of oils for component polyunsaturated acids, by this method, compare well with those by other methods. The presence of small amounts of a tetraenoic acid in linseed oil is noted. Postdoctoral Fellow in physiological chemistry. This work was supported in part from funds granted by the Ohio State University Research Foundation to the university for aid in fundamental research. Presented at the 31st Fall Meeting of the American Oil Chemists' Society, Cincinnati, O., September 30 to October 2, 1957.     

A rapid spectrophotometric method for determining the linoleic and linolenic acid components of soybean oil

Summary A reagent of 11% KOH-glycerol was satisfactory for isomerizing soybean oil at 180°C. in open bottles in a forced-draft air-oven. A rapid means of analyzing soybean oil for linolenic and linoleic acid was developed. The optical densities of methanol solutions were measured at 268 mμ and 233 mμ in special cells with quartz windows and short light paths, and the percentages were calculated by means of a nomograph. Publication No. 274 of the U. S. Regional Soybean Laboratory. Chemist and Agent (Physical Science Aid), respectively, Field Crops Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Analysis of linoleate and linolenate by isomerization with potassiumtertiary butoxide in mixtures oftertiary butanol and ethylene glycol dimethyl ether

A simple spectrophotometric procedure is described for determining the content of linoleic and linolenic acid in natural oils. Isomerization is accomplished at 70C with potassiumtertiary butoxide (0.57 M) in 110 min. The absorptivities of linoleic acid (k₂₃₃=86.9) and linolenic acid (k₂₃₃=66.9 and k₂₆₈=46.9) attain a maximum and remain constant thereafter.     

Studies on the chemistry of the fatty acids VIII the reaction of thiocyanogen with linoleic and linolenic acids

Summary The thiocyanogen reagent may be stabilized by storage at temperatures of 3° or less. The reaction of thiocyanogen with highly purified linoleic and linolenic acids has been studied under many variations of experimental conditions. Reaction rates of thiocyanogen with oleic, linoleic and linolenic acids at 3°, 16° and 25° and with variable excess of reagent are described. Oleic acid gives a maximum thiocyanogen absorption which is apparently equal to the iodine number, within 3–6 hours. The reaction with linoleic acid is rapid up to 3 hours and shows a slow constant increase thereafter. A modified procedure of analysis, similar to the official method, employs a 0.2 N thiocyanogen reagent, containing 10 percent carbon tetrachloride and a reaction temperature of 16°. Results of analysis of several specimens of acids by several preparations of reagent shows an average thiocyanogen value of 96.6 for linoleic acid and 166.3 for linolenic. Simultaneous equations, derived from these values, were used in the analysis of five mixtures of known composition with excellent results. Presented in part at the Cincinnati meeting of the American Chemical Society, 1940. Instructor in Biochemistry at Emory University, Emory University, Ga. Presented in partial fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University.     

Geometrical isomerization of polyunsaturated fatty acids in physically refined rapeseed oil during plant‐scale deodorization

The effect of the operating temperature (between 220 and 270 °C) on the formation of trans isomers of linoleic and linolenic acids in physically refined rapeseed oil during deodorization in a plant‐scale semicontinuous tray‐type deodorizer (capacity 10 t/h) was investigated. The industrial procedures of physical refining consisted of a two‐step bleaching and deodorization process. The degree of isomerization of linoleic acid ranged from 0.33 to 4.77% and that of linolenic acid from 4.43 to 45.22% between 220 and 270 °C, respectively. A relation between the logarithm of the degree of isomerization and the deodorization temperature can be approximated by statistically highly significant linear functions for both linoleic and linolenic acids. Oleic acid was resistant to the heat‐induced geometrical isomerization. The values found for the ratio between the degrees of isomerization of linolenic and linoleic acids, slightly decreasing with increasing temperature, were equal to 13.6 and 12.9 at 230 and 240 °C, respectively. Two trans isomers of linoleic acid, exclusively with one double bond isomerized into trans configuration, and four trans isomers of linolenic acid, mostly with one double bond isomerized into trans configuration, were determined in deodorized rapeseed oils. Linolenic acid was observed to be the main source responsible for the formation of nearly all trans fatty acids in physically refined rapeseed oil. At 235 °C, a deodorization temperature considered as a reasonable technological compromise, the content of trans fatty acids in plant‐scale physically refined rapeseed oil was less than 1% of total fatty acids, which would be acceptable for further application.     

Cis-trans isomerization of oleic,linoleic and linolenic acids

The equilibrium composition ofcis andtrans isomers obtained by isomerizing oleic, linoleic, and linolenic acids with selenium or nitrous acid has been studied using gas chromatography and infrared spectroscopy. The oleic/elaidic equilibrium mixture was found to contain 75–80% elaidic acid instead of the generally accepted 66% value. It is felt that the greater accuracy of gas chromatography and infrared analyses over older methods allows this equilibrium to be defined with greater precision. Similar studies on thecis-trans isomerization of linoleic and linolenic acids indicated that their equilibrium mixtures also contained 75–80%trans double bonds. With linoleic acid, thesetrans bonds were shown to be randomly distributed among the double bonds present. Cis-trans isomerization of linoleic or linolenic acids with selenium produced by-products having elution times equivalent to 18∶2, 18∶1, and 18∶0 on a gas chromatograph. No such by-products were observed when oleic acid was isomerized. Apparently some type of hydrogen-transfer reaction accompanies thecis-trans isomerization of polyunsaturated acids with selenium. Presented at the AOCS meeting in Toronto, Canada, 1962.     

Deodorization of vegetable oils. Part I: Modelling the geometrical isomerization of polyunsaturated fatty acids

Laboratory-scale treatments of canola oils similar to deodorization were carried out by applying the following conditions: reduced pressure with nitrogen or steam stripping at different temperatures ranging from 210 to 270°C for 2–65 h. The formation of the group of trans linolenic acid isomers follows a first-order reaction and the kinetic constant varies according to the Arrhenius’ law. Similar results were observed for the trans isomerization of linoleic acid. Based on these experiments, a mathematical model was developed to describe the isomerization reaction steps occurring in linoleic and linolenic acids during deodorization. The calculated degrees of isomerization are independent of the composition of the oil but related to both time and temperature of deodorization. The degree of isomerization of linolenic acid is unaffected by the decrease of this acid content observed during the deodorization. Deodorization at about 220–230°C appears to be a critical limit beyond which the linolenic isomerization increases very strongly. The newly established model can be a tool for manufacturers to reduce the total trans isomer content of refined oils, and was applied to produce a special selectively isomerized oil for a European Nutritional Project.     

Oxidative stability of conjugated linoleic acids relative to other polyunsaturated fatty acids

Contrary to current opinion, conjugated linoleic acids (CLA) as a mixture of several isomers have been previously shown to function as prooxidants in the form of free fatty acids and methyl esters in heated canola oil. Furthermore, CLA oxidizes considerably faster than linoleic acid. However, stability of CLA relative to other polyunsaturated fatty acids remains undetermined. The present study was therefore undertaken to examine the relative oxidation rate of CLA compared with that of linolenic acid (LNA), arachidonic acid (AA), and docosahexaenoic acid (DHA) in air at 90°C. CLA, both in the form of free fatty acids and triacylglycerols, were extremely unstable to the same extent as DHA, but they oxidized considerably faster than LNA and AA. The mechanism by which CLA were readily decomposed was probably due to formation of the unstable free-radical intermediate.     

trans-Polyunsaturated fatty acids in French edible rapeseed and soybean oils

The fatty acid compositions of rapeseed and soybean oils marketed in France have been determined by gas liquid chromatography on a fused-silica capillary column coated with a 100% cyanopropyl polysiloxane stationary phase. Under the operating conditions employed, methyl esters of linolenic acid geometrical isomers could be separated and quantitated easily without any other complementary technique. With only one exception, all samples under study (eight salad oils and five food samples) contain geometrical isomers of linolenic acid in measurable, although variable, amounts. Totaltrans-18:3 acids may account for up to 3% of total fatty acids. This value corresponds to a degree of isomerization (percentage oftrans isomers relative to total octadecatrienoic acids) of 30%. Examination of our data indicates that the distribution pattern of linolenic acid geometrical isomers does not depend on the degree of isomerization. The two main isomers always have thec,c,t and thet,c,c configurations. These isomers occur in the almost invariable relative proportions of 47.8±1.7% and 41.1±1.0%, respectively. The third mono-trans isomer is present in lower amounts−6.5±0.7%. The only di-trans isomer that can be quantitated with sufficient accuracy is thet,c,t isomer (4.9±1.5%). Mono-trans isomers of linoleic acid are also present in these oils. However, their maximum percentages are lower than those determined for linolenic acid geometrical isomers. In the oils showing the highest degrees of isomerization,trans isomers of linoleic acid account for 0.5% (rapeseed oils) and 1% (soybean oils) of total fatty acids. Taking into account all data, it would appear that the probability of isomerization of linolenic acid is about 13–14 times that of linoleic acid.     

The melting points of binary mixtures of oleic,linoleic, and linolenic acids

Summary 1. The melting points of binary mixtures of oleic, linoleic, and linolenic acids have been reported. 2. The oleic-linoleic acid system has eutectics for the α and β forms of oleic acid of 75.2 and 76.3 mole per cent linoleic acid, at −10.0° and −9.8°, respectively. 3. Linoleic and linolenic acid mixtures show only melting points intermediate between the pure acids. 4. The oleic-linolenic acid system has eutectics for the α and β forms of oleic acid of 82.7 and 85.5 mole per cent linolenic acid, at −15.7° and −15.1°, respectively. A cooperative organization participated in by the Bureaus of Agricultural Chemistry and Engineering and Plant Industry of the U. S. Department of Agriculture, and the Agricultural Experiment Stations of the North Central States of Illinois. Indiana, Iowa, Kansas. Michigan, Minnesota, Missouri, Nebraska, North Dakota, Ohio, South Dakota, and Wisconsin.     

Highly unsaturated fatty acids. III. Isolation of methyl eicosapentaenoate,ethyl docosapentaenoate,and ethyl docosahexaenoate from cod liver oil esters by chromatography

a) Displacement chromatography using a charcoal-isopropanol-methyl behenate system has been successfully applied to the isolation of docosahexaenoic acid and ethyl docosahexaenoate from cod liver oil concentrates. b) Methyl eicosapentaenoate was isolated from cod liver oil methyl esters by combined elution chromatography on silicic acid and displacement chromatography. c) Using silicic acid as adsorbent and petroleum ether-chloroform as solvent, ethyl docosapentaenoate was isolated from cod liver oil ethyl esters. d) With a spinning band fractionation column it was found that fractional distillation of methyl esters of cod liver oil at 0.5 mm. Hg. resulted in increasing conjugation in the distillate as the fractionation proceeded. Fractional distillation is to be avoided as a final step in purification of polyunsaturated esters. e) The end absorption of conjugated pentaenoic acids is so large at the characteristic maximum for hexaene (3,750 Å) that simultaneous equations are required for the calculation of hexaenoic and pentaenoic acids. f) Fifteen minutes of treatment with 21% KOH-ethylene glycol at 180° is not optimum for isomerization of ethyl docosapentaenoate from cod liver oil. The optimum isomerization times for hexaenoic acids of various sources are 4 to 6 minutes, considerably different from the 15 minutes established for arachidonate.     

Lipids and human milk

To support the growth and development of the breast‐fed infant, human milk provides the dietary essential fatty acids, linoleic acid (LA; 18:2n‐6), α‐linolenic acid (ALA, 18:3n‐3), as well as longer‐chain polyunsaturated fatty acids including arachidonic acid (20:4n‐6) and docosahexanoic (DHA 22:6n‐3). The linoleic acid, alpha‐linolenic acid, DHA and arachidonic acid concentration of pasteurized and unpasteurized human milk remains stable during the first month of storage at –20°C and –80°C. However after the first month, a slow decrease in concentration progresses until the end of 6 months of storage at both temperatures. The levels of n‐6 and n‐3 fatty acids, particularly linoleic acid, alpha‐linolenic acid and DHA, in human milk vary widely within and among different populations, and are readily changed by maternal dietary intake of the respective fatty acid. The present paper reviews recent understanding from key researchers of maternal diet and human milk fat composition and form our work the effect of milk fat composition on storage conditions. It is important to understand that maternal diet can affect human milk fat composition and subsequently infant development and growth.     

Application of urea complexes in the purification of fatty acids,esters, and alcohols. III. Concentrates of natural linoleic and linolenic acids

Summary Concentrates of natural linoleic acid (linoleic acid content, 85–95%) have been prepared in 50–72% yields from corn oil fatty acids by preferential precipitation of the saturated and monounsaturated fatty acids at room temperature as their urea complexes. By a similar procedure, concentrates of natural linolenic acid (linolenic acid content, 87–89%) have been prepared in 55–61% yields from perilla oil fatty acids by preferential precipitation of the saturated, monounsaturated, and diunsaturated fatty acids. Although concentrates of natural linolenic acid containing only 66–70% linolenic acid were obtained from linseed oil fatty acids, yields were 87–90%. A levelling-off effect has been observed in the use of the preferential precipitation technique in raising the purity of concentrates of linoleic and linolenic acid. This parallels the experience in the purification of these acids by low-temperature crystallization. The preceding papers in this series are references 12 and 13. Presented at the Fall Meeting of the American Oil Chemists' Society, Cincinnati, O., Oct. 20–22, 1952. One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, U. S. Department of Agriculture.     

Opportunities and problems in modification of levels of rapeseed C18 unsaturated fatty acids

Selections for different levels of C₁₈ fatty acids in rapeseed to date have had only limited success, due in part to the low frequency of occurrence of desired genotypes with increased linoleic and decreased linolenic acid. In the progeny of mutation experiments with seeds of the variety Oro (linolenic acid content 8–10%) two stable mutants were selected, one with 5% and the other with 20% linolenic acid in the seed oil. The level of linoleic acid in the two mutants is the same as Oro (16–20%), but the levels of oleic and linolenic acids are inversely altered. In this paper several problems associated with selecting for linoleic and linolenic acids, which became apparent during the mutation studies, are discussed. Many selections made from the mutated material were unstable, reverting to the original Oro fatty acid composition after two or three self-pollinated generations. This fact plus environmental and maternal effects made selection difficult. However, with the use of rapid and simple analytical methods and space-saving growing techniques, these difficulties were overcome. One of six papers presented in the symposium “Rapeseed Marketing and Breeding,” AOCS Meeting, Ottawa, September 1972.     

Deodorization of vegetable oils: Prediction of trans polyunsaturated fatty acid content

Kinetics of the formation of trans linoleic acid and trans linolenic acid were compared. Pilot plant-scale tests on canola oils were carried out to validate the laboratory-scale kinetic model of geometrical isomerization of polyunsaturated fatty acids described in our earlier publication. The reliability of the model was confirmed by statistical calculations. Formation of the individual trans linoleic and linolenic acids was studied, as well as the effect of the degree of isomerization on the distribution of the trans fatty acid isomers. Oil samples were deodorized at temperatures from 204 to 230°C from 2 to 86 h. Results showed an increase in the relative percentage of isomerized linolenic and linoleic acid with an increase in either the deodorization time or the temperature. The percentage of trans linoleic acid (compared to the total) after deodorization ranged from <1 to nearly 6%, whereas the percentage of trans linolenic acid ranged from <1 to >65%. Applying this model, the researchers determined the conditions required to produce a specially isomerized oil for a nutritional study. The practical applications of these trials are as follows: (i) the trans fatty acid level of refined oils can be predicted for given deodorization conditions, (ii) the conditions to meet increasingly strict consumer demands concerning the trans isomer content can be calculated, and (iii) the deodorizer design can be characterized by the deviation from the theoretical trans fatty acid content of the deodorized oil.     

The nature of the C18 polyethenoic fatty acids of butter fat

Methyl C₁₈ polyenoate concentrates were prepared from two samples of butter fat by low temperature crystallization and fractional distillation. The concentrates were fractionated on a silicic acid column and the resulting fractions were analyzed by ultraviolet and infrared spectrophotometric methods. About 42 and 30% repectively, of the non-conjugated dienoate in the two samples were shown to have thecis,trans configuration. Fractions rich in dienoate and trienoate were prepared from the C₁₈ polyene concentrates by silicic acid chromatography and the nature of these acids was studied by bromination, lipoxidase enzyme methods, and by alkali isomerization for varying periods of time. About 65 and 73%, respectively, of the non-conjugated dienoate in the two samples investigated were found to consist of linoleic acid while 79 and 71% of the trienoate were linolenic acid. Linoleic and linolenic acids were identified by preparing the characteristic tetra- and hexabromostearic acids. Atrans,trans isomer of linoleic acid does not seem to be present in butter fat. A major proportion of the non-conjugated dienoic acid other than linoleic acid was found to have widely separated double bonds withcis,trans configuration. Occurrences of a C₁₅ saturated acid, a branchedchain C₁₇ saturated acid, and a heptadecenoic acid were indicated by gas chromatography. From a dissertation submitted by K. Sambasivarao to the Ohio State University in partial fulfillment of the requirements for the Ph.D. degree, June, 1960. This work was supported in part by a grant from the Ohio State University Development Fund to the Institute of Nutrition and Food Technology and by several teaching assistantships from the Department of Physiological Chemistry.     

Comparative studies on composition of cardiac phospholipids in rats fed different vegetable oils

Male Sprague-Dawley rats were fed diets for 1 or 16 weeks, containing 20% by weight vegetable oils differing widely in their oleic, linoleic and linolenic acid content. No significant changes were observed in the level of the cardiac lipid classes. The fatty acid composition of the 2 major phospholipids, phosphatidylcholine and phosphatidylethanolamine, showed a remarkable similarity between diets in the concentration of total saturated, C22 polyunsaturated and arachidonic acids. Monounsaturated acids were incorporated depending on their dietary concentration, but the increases were moderate. Dietary linolenic acid rapidly substituted C22 polyunsaturated fatty acids of the linoleic acid family (n−6) with those from the linolenic acid family (n−3). The results suggest that dietary linolenic acid of less than 15% does not inhibit the conversion of linoleic to arachidonic acid but the subsequent conversion of arachidonic acid to the C22 polyunsaturates was greatly reduced. Significant amounts of dietary monounsaturated fatty acids were incorporated into cardiac cardiolipin accompanied by increases in polyunsaturated fatty acids, apparently to maintain an average of 2 double bonds/molecule. The cardiac sphingomyelins also accumulated monounsaturated fatty acids depending on the dietary concentration. It is quite evident from the results of this study that the incorporation of oleic acid and the substitution of linolenic for linoleic acid-derived C22 polyunsaturated fatty acids into cardiac phospholipids was related to the dietary concentration of these fatty acids and was not peculiar to any specific oil. Even though it is impossible to estimate the effect of such changes in cardiac phospholipids on membrane structure and function, results are discussed which suggest that the resultant membrane in the Sprague-Dawley male rat is more fragile, leading to greater cellular breakdown and focal necrosis. Contribution No. 914 from the Animal Research Institute.     

Oxidative stability of polyunsaturated fatty acids: effect of squalene

The propensity of polyunsaturated fatty acids (PUFAs) to undergo oxidation plays an important role in the integrity of biological membrane and lipid containing foods. The ability of squalene (SQ), a naturally occurring dehydrotriterpene present in animal and plant tissues, to protect linoleic, linolenic, arachidonic and docosahexaenoic acids against temperature‐dependent autoxidation and UVA (ultraviolet A, 320‐380 nm) mediated oxidation was assessed. The oxidation of PUFAs was protected in varying degrees, with highest protection observed for linolenic, arachidonic and docosahexaenoic acids. Linoleic acid was less protected. At a molar ratio of 7:1 (PUFA:SQ) the inhibition of the oxidation process was 22% in the presence of linoleic acid and about 50% in presence of the other PUFAs tested. The different protection exerted by SQ against PUFAs with different degrees of unsaturation may be accounted for by the higher stability of octadecadienoic acid hydroperoxide isomers compared with respective PUFA hydroperoxides. Observing mild UVA‐mediated oxidation and the temperature‐dependent autoxidation reactions we found similarities in the oxidation pattern and the protection exerted by SQ. These findings suggest that the reaction of autoxidation is predominant and SQ acts mainly as peroxyl radical scavenger.     

Effect of fatty acid positional distribution and triacylglycerol composition on lipid by-products formation during heat treatment: II. Trans isomers

This study examined the effect of the fatty acid positional distribution and of the triacylglycerol (TG) composition on heat-induced trans isomerization of linoleic and linolenic acids. For this, we synthesized diacid TG molecules that were acylated only with linoleic acid (L) or with linolenic acid (Ln) along with palmitic acid (P). The fatty acid of interest was positioned either in the central position (PLP and PLnP, respectively) or in one of the two outer positions (PPL and PPLn, respectively). Monoacid TG, i.e., trilinolein and trilinolenin, were also synthesized and mixed with tripalmitin in a 1:2 ratio. This model TG was also compared to another TG model, which consisted of a canola oil and its randomized counterpart whose fatty acid positional distribution and TG composition were determined by means of high-performance liquid chromatography. After heating, the content of trans isomers was determined by gas-liquid chromatography with a polar capillary column. In model TG, polyunsaturated fatty acids in monoacid TG (LLL and LnLnLn) exhibited the highest degree of isomerization, compared to diacid TG, and this effect was greatest at 220°C. At this temperature, an effect of the TG structure was observed only with linolenic acid. In that situation, 18:3n-3 acylated in the central position of the TG molecule (PLnP) displayed the highest sensitivity to trans geometrical isomerization. Although to a lesser extent, the same trends as for the pure TG model were observed with the canola oil model with regard to the influence of the fatty acid positional distribution and TG molecular species.