Effects of ingredients on the oxidation of linoleic acid by lipoxygenase in bread doughs

Flour and water doughs containing 1-¹⁴C-linoleic acid (18:2) and various ingredients were prepared to study the oxidation of linoleic acid by lipoxygenase in bread doughs. Lipids were extracted, treated with diazomethane, and ¹⁴C-labelled fatty acid methyl esters separated by thin-layer chromatography. Radioactivity was determined in silica gel bands containing unoxidised 18:2, hydroperoxy acids (L₁), hydroxy acids (L₂), hydroxyepoxy acids (L₃) and trihydroxy acids (L₄). Minor components detected by autoradiography were present mainly in L₃ and L₄. Recoveries of total radioactivity were always > 95%. Untreated flour-water dough was mixed aerobically for ?4 min, rested, and the lipids extracted after 10 min total dough time. Yields of ¹⁴C products were unoxidised 18:2 = 28.6 μmol, L₄ = 93.9 μmol/100 g dry flour. Similar yields were obtained from ClO₂-treated flour, both after 10 min and 60 min dough time. Salt, salt + yeast, or salt + yeast + ascorbic acid in the dough did not reduce 18:2 oxidation significantly, but increased L₃ at the expense of L₄. Soya flour preparations inhibited linoleic acid oxidation by 25–44%, but pure soya lipoxygenase had no effect at all. Heat treatment reduced the inhibitory effect of soya flour. Accessible thiol groups were not essential for lipoxygenase activity or for the reduction of L₁ to L₂ since adding cysteine or N-ethyl maleimide had negligible effects on the 18:2 oxidation products. Most of the flour carotenoids (xanthophylls) were bleached by wheat enzymes in non supplemented doughs, and all were bleached in doughs supplemented with soya flour. ¹⁴C-labelled triglyceride was not oxidised except in doughs containing soya flour mixed in air (1.5% oxidation) or oxygen (3 % oxidation). Soya flour contains lipoxygenase isoenzymes (principally lipoxygenase-2) which oxidise linoleate in triglycerides. This isoenzyme is evidently not present in wheat.     

Changes in wheat lipids during mixing and resting of flour-water doughs

Lipids extracted with water-saturated n-butanol from flour and flour-water doughs were examined to determine the extent of oxidations and other changes which occurred in mixing and resting dough. Extracted lipids were converted to fatty acid methyl esters (FAME) and quantified by gas-liquid chromatography (g.l.c.) using heptadecanoic acid (17 : 0) as internal standard. The original flour or dough and the corresponding solvent-extracted residues were acid hydrolysed, and the hydrolysate lipids converted to FAME for g.l.c. determination of the total lipid and residual unextracted lipid contents. The total flour or dough lipids equalled the extracted lipids + unextracted lipids, except where there were unavoidable autoxidative losses of linoleate (18 : 2) and linolenate (18 : 3). The unextracted flour lipids (13% of total lipids) were not oxidised during dough mixing. There were no changes in any of the extracted lipid classes other than free fatty acids (FFA) and monoglycerides (MG) which showed losses of 18 : 2 and 18 : 3 after aerobic dough mixing. Losses of FFA and MG are attributed to lipoxygenase activity during dough mixing and the period immediately after. The small amount of 18 : 2 in the “free” petroleum ether-extracted FFA appeared to be unaffected by lipoxygenase. Recoveries of FFA other than 18 : 2 or 18 : 3 were constant, indicating no lipolysis of glycerolipids and no general oxidation or degradation of FFA. Experiments with [U-¹⁴C]palmitic acid confirmed that there was no oxidation, degradation or re-esterification of FFA. Much of the non-polar lipids (steryl ester, triglyceride, diglyceride, FFA, MG) and almost all of the polar lipids were bound on dough mixing. Binding was non-selective with regard to fatty acid composition. Triglyceride was the only lipid class bound to a greater extent in anaerobic dough than in aerobic dough. Some selectivity of binding between lipid classes was indicated.     

The effect of lipoxygenase action on the mechanical development of doughs from fat-extracted and reconstituted wheat flours

The mechanism by which soya lipoxygenase enzyme action improves the Theological properties of wheat flour doughs during mechanical development in air has been investigated further. Free-lipid extraction, reconstitution and replacement experiments have shown that the rheological effect of lipoxygenase action, which is consistent with an oxidative improvement of the dough proteins and may also result in extended mixing tolerance, only occurred in the presence of an oxidisable, polyunsaturated, free-lipid substrate. Addition of this substrate in an oxidised state (produced either by autoxidation or enzyme-oxidation) to doughs mixed from fat-extracted flour under nitrogen resulted only in a small rheological improvement, greater for the autoxidised than the enzyme-oxidised lipid, but in no way comparable with the large rheological effect of lipoxygenase action during dough mixing in air. Furthermore, the presence of an antioxidant, nordihydroguaiaretic acid (NDGA), during dough development, although greatly inhibiting peroxide formation, only marginally impaired the rheological improvement due to lipoxygenase action. Additional evidence is therefore provided for a coupled oxidation mechanism being responsible for the rheological effect, since lipoxygenase-catalysed oxidation actively occurring in the dough during mixing appears to be the fundamental requirement, irrespective of whether the primary oxidation products lead to lipid peroxides or oxidised NDGA.     

The effect of lipoxygenase action on the mechanical development of wheat flour doughs

The mechanical development of dough has been followed by measurement of the relaxation time of dough samples after mixing to various levels of work input. Parallel determinations of free and bound lipid have also been made. When doughs were mixed in air, addition of full-fat, enzyme-active soya bean flour (subsequently referred to as “soya flour”) resulted in an increase in relaxation time, particularly at higher work levels. The magnitude of this improvement increased with increasing work input and was dependent on the rate of work input. Addition of soya flour also enabled a higher level of mechanical work to be introduced before dough breakdown occurred. When doughs were mixed under nitrogen, or when the soya flour was heat denatured, no change in the rheological properties compared with the respective control doughs was found. The release of bound lipid, which occurred during dough development in air in the presence of soya flour, could also be induced by adding purified lipoxygenase to the dough, together with linoleic acid as a substrate. This resulted in rheological changes similar to those observed using soya flour. However addition of enzymically pre-peroxidised lipid to doughs mixed in nitrogen was without effect on relaxation times. These findings suggest that lipid release is linked with structural changes in dough protein and provide further support for a mechanism of coupled oxidation of protein -SH groups by lipoxygenase.     

Changes in flour lipids during the storage of wheat flour

High- and low-grade spring and winter wheat flours of ～13% moisture were stored at 15, 25 and 37 °C and the lipids were then extracted with water-saturated n-butanol. In the original (control) flours there were more neutral lipids and glycolipids in low-grade winter than in high-grade winter and in low-grade spring than in high-grade spring flours, but there were no corresponding differences in the amounts of phospholipids. The total extractable lipid contents of the flours remained constant in the samples stored at 15 °C, but there were slight losses in the samples stored at 25 and 37 °C. Total lipid contents determined by acid hydrolysis remained constant in all cases indicating that no loss of fatty acids had occurred on storage. There was sufficient hydrolysis of all glycerides to account for the increased amounts of free fatty acids in the stored flours. Some complete deacylation of lipids to free fatty acids and water-soluble products was indicated. The fatty acid composition of all lipids remained constant, and there was no evidence of any lipoxygenase or other enzymic degradation of fatty acids. Stereoanalysis of the principal glycerides indicated that phosphatidylcholine (and probably also phosphatidylethanolamine) was specifically hydrolysed at the 2-position, presumably by phospholipase-A₂. Hydrolysis of triglycerides, diglycerides and monoglycerides was attributed to the action of wheat and microbial lipases of unknown specificity. Stereoanalysis of N-acylphosphatidylethanolamine and the galactosyldiglycerides was not attempted, but it was deduced that they were randomly hydrolysed at the 1- and 2-positions. The changes found in the flour lipids differed from those reported to occur in germinating wheat and in stored damp wheat flour which had been damaged by moulds.     

Changes in lipid components during the development of the french bean seed (Phaseolus vulgaris)

Changes in lipid classes, fatty acid composition and distribution have been monitored during the germination, development and maturation of the French bean seed Phaseolus vulgaris. A lipase activity profile over time was also determined. Ungerminated seeds contained high levels of triglycerides and free fatty acids, but low levels of polar lipids, monoglycerides, and diglycerides. Five days after germination there was a decrease in the quantity of triglycerides and free fatty acids and a concomitant increase in the levels of monoglycerides, diglycerides, and polar lipids. As development and maturation progressed, triglycerides increased substantially at the expense of mono- and diglycerides. Ungerminated seeds contained high levels of C₂₀-C₂₂ fatty acids which decreased after germination with a concomitant increase in C₁₆-C₁₈ unsaturated fatty acids. A study of the fatty acid distribution among the different classes of lipids demonstrated that 55% of the unsaturated fatty acids in the ungerminated seeds were present in the triglycerides, whereas the remainder were distributed among the mono- and diglycerides as well the other lipid classes (free fatty acids, polar lipid, and sterols). Five days after germination, the majority of these unsaturated fatty acids were found in the glyceride form. Overall the lipid classes, fatty acid composition and distribution changes during development and maturation of the French bean seed indicated that it shares many of the characteristics of soya bean development.     

Metabolism of citric and malic acids during ripening of tomato fruit

Tomato fruit at various stages of ripeness were injected with ¹⁴C-labelled citric and malic acids. The ¹⁴CO₂ evolved by individual fruit was determined over a 72 h period and the fruit then analysed for the incorporation of ¹⁴C into sugars, amino acids and individual non-volatile organic acids. In mature green tomato fruit citric and malic acids were found to be metabolised to a comparable extent. Appreciable interconversion occurred together with oxidation to CO₂ and some labelling of glutamic acid and glutamine. In red fruit, citric acid was not as readily oxidised to CO₂ and little conversion to malic acid was observed. On the other hand, malic acid was oxidised to CO₂ and converted to citric acid as rapidly as in green fruit with, in addition, appreciable conversion to glutamic acid. These results are discussed in relation to the changes in concentration of the endogenous acids which take place during the ripening of tomato fruit.     

Identification and quantitative determination of the lipids of dried Origanum dictamnus leaves

The Origanum dictamnus plant was examined for its lipid and fatty acid compositions. A combination of chromatographic techniques has been employed for the qualitative and quantitative determination of the lipids of dried leaves of O. dictamnus. The following polar lipids were identified: mono-, di- and poly-digalactosyl diglycerides, sulpholipids, cerebrosides, phosphatidyl-ethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, phosphatidyl choline and phosphatidic acid. The non-polar lipids identified were sterols, steryl esters, fatty alcohols, free fatty acid, waxes, traces of triglyceride, triterpenic acids and essential oil. The predominant fatty acids were palmitic, oleic and linoleic acids.     

Composition of Ghanian fermented maize meal and the effect of soya fortification on sensory properties

There were no significant differences in the composition of four fermented maize doughs used by different ethnic groups in Ghana. The average maize dough contained 49.6% moisture, 37.5% starch and had a titratable acidity of 4.3 mg NaOH g^?1 and pH of 3.76. Fermented maize dough (48% m.c.) was fortified with defatted soya flour at 50, 100, 150 and 200 g kg^?1 on a replacement basis and used in two traditional food products. Triangle taste tests indicated a significant difference between the control and samples fortified with 10% or more soya flour. Based on a preference test, maize dough products with up to 10% added soya flour were acceptable to Ghanaians.     

The use of chromic oxide as a food marker —a warning

Four mixtures of ground barley and soya bean meal were prepared; the first had no further additive, the second had 10% soya bean oil, the third 0.05% chromic oxide and the fourth both 10% soya bean oil and 0.05% chromic oxide. Each of these mixtures was then dried in three ways: at 30°C in vacuo and at 60 and 100°C in a forceddraught oven. The total fatty acid content and the fatty acid composition of each sample was determined either by direct esterification of the fatty acids with methanolsulphuric acid or by extraction into chloroform-methanol followed by esterification with sodium methoxide. When the mixture containing both oil and chromic oxide was heated at 100°C, about 25% of the polyunsaturated fatty acids were lost when they were directly esterified and about 50% when the fat was first extracted from the mixture. It is concluded that this loss is probably due to oxidation of the polyunsaturated fatty acids by the chromic oxide and that the additional loss observed when the extraction method was used is due to partial insolubilisation of the triglyceride by these oxidised fatty acids.     

The lipids of young cassava (Manihot esculenta,Crantz) leaves

Lipids were extracted quantitatively from young cassava (Manihot esculenta Crantz) leaves with a chloroform-methanol mixture. Total lipids were purified by the Folch procedure and separated into non-polar lipid, glycolipid and phospholipid fractions by column chromatography. Lipids of each fraction were further subjected to thin layer chromatography and gas-liquid chromatography. Young cassava leaves were found to have low content of lipids (3.02%) of which 22.4, 25.1 and 48.2 were non-polar lipids, glycolipids and phospholipids, respectively. Pigments (11.5%), wax and hydrocarbons (1.2%), steryl esters (2.9%), methyl esters of fatty acids (2.0%), trigly-cerides (1.5%), fatty acids (2.1%), diglycerides (1.1%) and sterols (0.1%) constituted the leaf non-polar lipids. The leaf glycolipids were made up of esterified steryl glycosides (2.1%), monogalactosyl diglycerides (12.5%), steryl glycosides (1.1%), cerebrosides (4.2%) and digalactosyl diglycerides (5.2%). The leaf phospholipids were found to include cardiolipin (3.6%), phosphatidylglycerol (21.5%), phosphatidylethanolamine (16.4%), phosphatidylserine (0.7%), phosphatidylinositol (4.0%) and other unidentified phospholipids (2.5%). Phosphatidylcholine was present only in trace quantity. Analysis of the fatty acid composition of each of the leaf lipids revealed that, with the exception of steryl esters, all leaf lipids have high content of polyunsaturated fatty acids.     

Short communication: Ability of cultured mammary epithelial cells in a bicameral system to secrete milk fat

Mammary epithelial cells from lactating cows were cultured onto inserts coated with type I collagen. Every second day, the rates of fatty acid synthesis and secretion were determined by measuring the amount of [¹⁴C]-labeled sodium acetate incorporated into lipids over a 4-h period. The [¹⁴C]-containing lipids were identified by thin layer chromatography fractionation. In parallel, the integrity of the cell layer was evaluated by measurement of transepithelial electrical resistance. The integrity increased progressively to reach a maximum after 8 d of culture. Cells incorporated acetate into lipids; 1.34% of acetate was incorporated into lipids produced by freshly isolated cells. This percentage decreased to 0.5% after 2 d of culture. Moreover, this capacity decreased with the duration in culture; on d 8, the rate of incorporation dropped to about 3% of that on d 2. In the cell extracts, the [¹⁴C]-labeled lipids were mainly triglycerides, although the proportion of diglycerides and phospholipids progressively increased as a part of total newly synthesized lipids. The proportion of triglycerides decreased 0.66 times between d 2 and 8 when the proportion of diglycerides and phospholipids increased 1.33 and 2.18 times, respectively. About 28% of the newly synthesized lipids were secreted within 4 h of incubation. Around 65 to 85% of these labeled lipids were found in the apical compartment, suggesting a partially vectorial secretion. But 58 to 80% of labeled lipids found in the apical and basolateral medium were free fatty acids. Functional tight junctions and incorporation of labeled fatty acids into triglycerides are not compatible with an inferred status of complete dedifferentiation of the cell layer. Moreover, triglyceride secretion seems compromised, probably due to the lack of an appropriate cell environment and cell shape.     

Mesocarp,seed and pollen lipids of Raphia palms

The lipid classes, fatty acids and sterols of the mesocarp, seed and pollen lipids of five species of Raphia palms endemic to Nigeria were analysed. Apart from quantitative differences in the fatty acid compositions, chromatographic analyses demonstrated very little change in the patterns of the characteristic lipids associated with either the mesocarps, seed endosperms or pollens. Mesocarp lipids contained mainly triglycerides, while palmitic, oleic and linoleic acids were the major acids. Contrary to published data for seed lipids, Raphia seed endosperms contained a relatively small amount of triglycerides and a high proportion of polar lipids. Fatty acids associated with this tissue were not only highly unsaturated, but were significantly different from typical fatty acids of seed lipids. Raphia pollens, on the other hand, showed a complex array of lipid types. Triglycerides, sterols and sterol esters constituted the main neutral lipids while phosphatidyl choline, phosphatidyl inositol, phosphatidyl ethanolamine and galactosyl diglycerides represented the polar fraction. Apart from minor trends, palmitic, oleic and linoleic acids constituted the major fatty acids in all species. The biological importance of the steroidal sapogenins found in all organs is discussed.     

Changes in the lipid and fatty acid composition during maturation of seeds of white mustard (Sinapis alba)

The oil content of the seeds of white mustard (Sinapis alba) was determined 3 weeks after flowering and at weekly intervals until seeds were matured. Examination of the lipid classes by quantitative t.l.c. showed that triglycerides were the major components at all stages and were accompanied by sterol esters, diglycerides and polar lipids; monoglycerides were only detected in the first 2 samples. The fatty acid composition of the total oil and the mono-, di- and triglycerides and sterol esters was determined by g.l.c. The 7 major fatty acids, namely palmitic, stearic, oleic, linoleic, linolenic, eicosenoic and erucic acids were present at all stages of maturity. The fatty acid composition of the triglyceride fraction was similar to that of the total oil whereas the mono- and diglycerides and sterol esters contained a relatively higher proportion of saturated fatty acids. Erucic acid, the major acid of the oil from mature seeds did not become the major acid until 35 days after flowering. The results are discussed in relation to pathways of lipid synthesis in higher plants.     

Genotype,environment and genotype × environment effects on trypsin inhibitor in peas

Four multiparous ruminally canulated cows and four primiparous cows without a ruminal cannula were used in a 4 × 4 Latin square experiment to study the effect of dietary fat source on milk fatty acid composition. Cows were fed a diet containing 550 g lucerne silage and 450 g concentrate kg^?1 dietary DM. The four diets were control and fat supplementation (32 g fat kg^?1 diet DM) as roasted soya beans, calcium salts of palm oil fatty acids (Megalac®). or hydrogenated tallow (Alifet®). Cows fed supplemental fat as roasted soya beans. Megalac®. or Alifet® produced 33.8, 32.6, and 32.5 kg day^?1 of 3.5% FCM. respectively, compared with 31.6 kg day^?1 with no fat supplementation. Addition of fat decreased milk protein percentage. Milk fat percentage was increased with Megalac® and decreased with Alifet® feeding. Dietary fat decreased the proportion of short- and medium-chain fatty acids (C6-C14:0) in milk and increased the proportion of long-chain fatty acids (C16 : 0 and longer). Roasted soya beans were most effective in reducing C16 : 0 and increasing C18 : 1, C18 : 2 and C18 : 3 acids in milk fat. Megalac® did not decrease milk C16 : 0 content, and neither Megalac® nor Alifet® affected C18 : 2 content.     

Effects of binary organic solvents and heating on lipid removal and the reduction of beany odour in Bambara groundnut (Vigna subterranean) flour

Effects of various binary organic solvents at different temperatures on the removal of lipids and beany or grassy odour of Bambara groundnut flour were studied. The highest lipid removal was achieved at 60 °C (P < 0.05), regardless of binary organic solvents used. Under the optimal temperature, chloroform/methanol showed the highest lipid removal (87%), followed by hexane/isopropanol (78%). All binary solvents containing methanol had higher efficiency in removal of phospholipids, and inactivation of lipoxygenase and trypsin inhibitors, as compared to isopropanol containing solvents (P < 0.05). Based on FTIR spectra, lipids removed by methanol containing solvents had high content of phospholipids. The flours defatted by methanol containing solvents exhibited the lowest peroxide value, thiobarbituric acid-reactive substances and beany odour intensity than the non-defatted flour and those defatted by isopropanol containing solvents throughout the storage (P < 0.05) of 30 days at refrigerated and room temperatures. In general, chloroform/methanol was the most effective in inactivating lipoxygenase and trypsin inhibitors, retarding lipid oxidation as well as beany odour development in flour. Therefore, chloroform/methanol could be used to lower beany or grassy odour in Bambara groundnut flour.     

The enzymic breakdown of lipids to volatile and non-volatile carbonyl fragments in disrupted tomato fruits

Physical disruption of tomato fruits results in the degradation of endogenous lipids by hydrolytic and oxidative enzymes. Acyl hydrolase, phospholipase D, lipoxygenase and hydroperoxide cleavage enzymes are active in this tissue. A sequential enzymic pathway is proposed by which tissue lipids are hydrolysed to free (mainly polyunsaturated C₁₈) fatty acids which are subsequently oxidised to their hydroperoxides by lipoxygenase action. The C₁₈ hydroperoxides are cleaved (by an enzyme which is specific for 13-hydroperoxide isomers) to volatile C₆ aldehydes (hexanal or cis-3 hexenal) and non-volatile C₁₂ w-oxoacids. The non-volatile fragment from linoleic acid was identified as 12-oxo-dodec-cis 9-enoic acid.     

Selective extraction and quantitative analysis of non-starch and starch lipids from wheat flour.

Wheat flour non-starch lipids (lipids not bound to starch) were quantitatively extracted with water-saturated n-butanol (WSB), benzene-ethanol-water (10:10:1, by vol.) or ethanol-diethyl ether-water (2:2:1, by vol.) in 10min at 20 °C. Starch lipids (lipids bound to starch) were subsequently extracted with WSB at 90–100 °C. Carotenoid pigments were quantitatively extracted with the non-starch lipids. There was no significant hydrolysis of esterified fatty acids and no detectable autoxidation of unsaturated acids in the hot solvent extracts. Non-starch and starch lipids from a high grade spring wheat flour and three grades of winter wheat flour were separated by thin-layer chromatography and quantified as fatty acid methyl esters (FAME) by gas-liquid chromatography (g.l.c.) using heptadecanoic acid (or its methyl ester) as internal standard. Total flour and starch lipids after acid hydrolysis were also converted to FAME for quantitation by g.l.c. Non-starch lipids consisted of 59–63% non-polar (neutral) lipids, 22–27% polar glycolipids and 13–16% phospholipids. Steryl esters, triglycerides, and all the diacyl galactosylglycerides and phosphoglycerides were present only in non-starch lipids. Starch lipids consisted of 6–9% non-polar (neutral) lipids (mostly free fatty acids), 3–5 % polar glycolipids and 86–91 % phospholipids (mostly lysophosphatidyl choline). Starch lipids were almost exclusively monoacyl lipids. Factors are given for converting weight of FAME into weight of original lipid for all individual lipid classes in wheat which contain O-acyl groups. Factors for total lipids are: total starch lipids = FAME × 1.70, total non-starch lipids = FAME × 1.20, and total flour (non-starch + starch) lipids = FAME × 1.32. Similar factors could be used to convert weight of lipids obtained by conventional acid hydrolysis methods into weight of unhydrolysed lipids. Phospholipid contents are given by: total starch phospholipids = P × 16.51, total non-starch phospholipids = P × 23.90, total flour phospholipids = P × 17.91.     

Effects of different levels of trans fatty acids and oxidised lipids in diet on cholesterol and cholesterol oxidation products formation in rabbit

The objective was to assess the effects of trans fatty acids and oxidised lipids, present in dietary fat by-products used in feeds, on cholesterol and oxycholesterols in meat, liver and plasma of rabbits. A palm fatty acid distillate, before and after hydrogenation (trans fatty acid trial), and a sunflower–olive oil blend (70/30, v/v), before and after use in a commercial frying process (oxidised lipid trial), were used in experimental feeds (at 3%, w/w). High trans fatty acid and oxidised lipid diets caused significantly higher cholesterol and oxycholesterol levels in all tissues of rabbit (0.01 < p ? 0.05). The content of oxycholesterols in rabbit meat, liver and plasma obtained from trans fatty acid experiments varied from 9 to 34 μg/100 g, 24 to 61 μg/100 g and 60 to 138 μg/dl, respectively, from low to high levels. In the oxidised lipid experiments, content of oxycholesterols varied from 16 to 52 μg/100 g, 14 to 108 μg/100 g and 52 to 269 μg/dl in meat, liver and plasma, respectively. As a consequence, meat products from rabbits fed a diet containing higher levels of trans fatty acids or oxidised lipids may result in higher intakes of cholesterol and oxycholesterols by humans.     

Reactions of Aqueous Chlorine and Chlorine Dioxide with Lipids: Chlorine Incorporation

Various lipids were reacted with HO³⁶Cl and ³⁶ClO₂ in an aqueous medium, and the extent of incorporation of ³⁶Cl into the lipids was determined. Cl incorporation into lipids treated with HOCl was greater than lipids treated with ClO₂. Free fatty acids incorporated more Cl than their corresponding esters. The degree of Cl incorporation was directly correlated with number of double bonds in the lipid. Triglycerides behaved as esters of their component fatty acids. High Performance Liquid Chromatography (HPLC) studies indicated at least two chlorinated products were formed from the reaction of HO³⁶Cl with oleic acid.