The Di- and triesters of the lipids of steer and human meibomian glands

Three groups of diesters have been isolated and identified in the lipids of steer meibomian glands. The first group, designated as α Type I, with the abbreviated formula FA-αOHFA-FA1c, consisted of α-hydroxy fatty acids esterified to fatty acids and fatty alcohols in the approximate molar ratio 1∶1∶1. The second group, designated as ω Type I-St, with the abbreviated formula FA-ωOHF A-St, consisted of ω-hydroxy fatty acids esterified to fatty acids and sterols in the approximate molar ratio 1∶1∶1. The third group, designated as α,ω Type II, with the abbreviated formula FA-α,ωdiol-FA, consisted of α,ω-diols esterified to 2 moles of fatty acids. The sum of the different diesters comprised about 9% of total steer meibomian lipids. Capillary GLC of the fatty acids of αType I diesters showed the fatty acids to be a family with a two-cluster profile, one at C₁₂ to C₂₀ and the other at C₂₁ to C₃₁, with anteiso chains predominating. Fatty acids from ωType I-St and α,ωType II diesters gave mainly a one-cluster profile in the short long chain, C₂₃ to C₃₀, with anteiso chains predominating, while the α-hydroxy fatty acids were short chain C₁₃ to C₁₈ acids with C₁₆ predominating. The sterols in diesters ωType I-St were cholesterol (∼60%), Δ7 cholestenol (∼35%) and an unidentified compound (∼5%) with a GLC retention time slightly longer than Δ7 cholestenol on SE-30 phase. The ω-hydroxy fatty acids and α,ω-diols both were of exceedingly long chain lengths, C₂₉−C₃₈, and showed similar GLC profiles. Two types of triesters comprising approximately 1% of total steer meibomian lipids have been isolated but incompletely characterized. In terms of molar ratios, one group of triesters gave fatty acids:ω-hydroxy fatty acids:α-hydroxy fatty acids:sterols + fatty alcohols as approximately 1∶1∶1∶1. The other contained fatty acids, α-hydroxy fatty acids and α,ω-diols in what appears to be a complex mixture of several triesters. Diesters ωType I and α,ωType II also were found in human meibum. Hitherto these two diesters have not been found in any animal tissue.     

Diester waxes in surface lipids of animal skin

The literature is surveyed on two types of diester lipid that occur on the skin surfaces of animals: Type 1, a hydroxy fatty acid of which the hydroxyl group and the carboxyl group are esterified respectively with another fatty acid and a fatty alcohol, and Type 2, and alkane α,β-diol of which each OH group is esterified with a fatty acid. New data presented here show that the cow, rabbit and cat produce Type 1, whereas the dog, mouse, guinea pig and baboon produce Type 2 diesters. Each occurs as a major component of the surface lipid. The homologue distribution is given for Type 1 diesters of cow, rabbit and cat as well as the Type 2 diesters of dog and mouse. Distribution of long chain fatty acids of Type 1 diesters parallels that of the fatty alcohols suggesting a biogenetic relation between the two types of compounds. GLC of total diesters for the cow suggests that the components are assembled randomly during biosynthesis. Molecular weight of these diesters are in the range of those of natural triglycerides composed mainly of C₁₆ and C₁₈ fatty acids. Presented at the 60th AOCS Annual Meeting, San Francisco, April 1969, as part of a Symposium on Natural Waxes.     

Comparative study of the molecular species of chloropropanediol diesters and triacylglycerols in milk fat

In an effort to establish the origin of the fatty acid esters of 3-chloropropanediol, which recently have been isolated in small amounts from goat milk, we compared the molecular species composition of the chlorohydrin diesters and of goat milk triacylglycerols. The chloropropanediol diesters were found to be composed of molecular species containing C₁₀−C₁₈ fatty acids and corresponded closely in carbon number to those calculated for the long chain sn-1,2-diacyl-glycerol moieties of goat milk triacylglycerols. The molecular species of goat milk total triacylglycerols contained C₄−C₁₈ fatty acids. It is suggested that triacylglycerols and chloropropanediol diesters are derived from the same pool of long chain fatty acids. A molecular distillate of bovine milk fat did not contain chloropropanediol diesters, while the available samples of human milk fat were shown to contain alkyldiacylglycerols as the major components of a neutral lipid fraction corresponding in polarity to the chloropropanediol diesters.     

Skin surface lipids of the dog

The skin surface lipid of the dog has been reported to contain a high proportion of diol diesters having a lower mobility on thin layer chromatography than diesters from other species in spite of containing similar fatty acid and diol components. In the present study, dog skin surface lipid was separated by preparative thin layer chromatography into sterol esters (42%), wax diesters (32%), free sterols (9%), polar lipids (7%), and unidentified components (10%). The diesters contained 1,2-diols, each esterified with one long chain fatty acid and one isovaleric acid moiety. The diols were principally branched chain C₂₁ and C₂₂ compounds while the long chain fatty acids esterified with them were mainly C₂₀ and C₂₁ branched compounds. The fatty acids from the sterol esters were mostly saturated, branched chain C₁₉ to C₂₃, together with 7% of straight chain monoenoic acids, principally C₂₁ and C₂₂. There were only trace amounts of free sterols other than cholesterol, while the esterified sterols contained 96% cholesterol and 4% lathosterol.     

The positional distribution of fatty acids in the phospholipids and triglycerides ofMycobacterium smegmatis andM. bovis BCG

Position 1 of the phospholipid and triglyceride fractions isolated fromMycobacterium smegmatis andM. bovis BCG was esterified principally with C₁₈ related fatty acids (18∶0, 18∶1 and 19Br). Position 2 was occupied principally by C₁₆ fatty acids. The third position of the triglycerides was esterified with a preponderance of C₂₀+fatty acids. Seventysix per cent of position 3 fatty acids in BCG and 43% inM. smegmatis triglycerides contained fatty acids of greater than 20 carbon atoms.     

Milk fat structure of a patient with type 1 hyperlipidemia

Structural analyses were performed on milk fat samples obtained 3–10 days postpartum from a lactating patient with primary Type 1 hyperlipidemia. The milk triacylglycerols contained 3–7% C₁₀, 14–21% C₁₂, 20–30% C₁₄, 22–26% C₁₆ and 20–30% C₁₈ (largely oleic) acids. Gas liquid chromatographic (GLC) analyses of the X-1,3- and X-1,2-diacylglycerols on polar siloxane columns showed a markedly non-random association of acyl chains. Stereospecific analyses indicated that the short chain length fatty acids were confined essentially to the sn-3-position of the triacylglycerol molecule. Furthermore, these acids were largely absent from the phosphatidylcholines and the endogenous sn-1,2-diacylglycerols of the milk fat. It is concluded that the short chain fatty acids are incorporated into the milk triacylglycerols during the final stage of biosynthesis via the phosphatidic acid pathway, and that the overall fatty acid distribution is consistent with the 1-random 2-random 3-random hypothesis.     

Fatty acids of the alkane diol diesters of vernix caseosa

The fatty acid monoenes esterified to the alkane diol diesters of vernix caseosa lipid form two patterns of homologues starting from either C₁₆Δ9 or C₁₆Δ6 and adding (or subtracting) an integral number of C₂ units at the carboxyl group. Although components of the Δ6 pattern are the predominant monoenes of sebaceous gland ester lipid classes, for these diol diesters Δ9 pattern components are preferentially used.     

Fatty chain compositon of ether and ester glycerophospholipids in the Japanese oysterCrassostrea gigas (Thunberg)

The fatty chain compositions of 1-O-alk-1′-enyl-2-acyl, 1-0-alkyl-2-acyl, and 1,2-diacyl glycerophospholipids of the Japanese oysterCrassostrea gigas (Thunberg) were investigated. Major fatty chains in thesn-1 position of 1-alk-1′-enyl-2-acyl ethanolamine phospholipids (EPL) were 18∶0 (64.7%) and 20∶1 (11.1%). Majorsn-1 chains of alkenylacyl choline phospholipids (CPL) were 18∶0 (63.3%) and 16∶0 (22.2%). In the case of 1-alkyl-2-acyl EPL, the predominant fatty chains in thesn-1 position were 18∶0 (51.5%), 16∶0 (16.0%) and 20∶1 (12.5%); in the case of 1-alkyl-2-acyl CPL, the majorsn-1 chains were 16∶0 (44.0%) and 14∶0 (23.4%). Saturated fatty chains were predominant in both EPL and CPL. Prominent fatty acids in thesn-2 position of the alkenylacyl EPL were 22∶6n−3 (29.0%), 20∶5n−3 (19.0%) and 22∶2 NMID (non-methylene interrupted dienes, 16.6%) contributing to about 65% of the total fatty acids, while alkenylacyl CPL was rich in the saturated acids 16∶0 (32.0%) and 18∶0 (9.2%). In the alkylacyl EPL, 16∶0, 18∶1n−9, 18∶0 and 16∶1n−7 were prominentsn-2 fatty acids and accounted for 30.6%, 10.0%, 9.8%, and 8.3%, respectively. Polyunsaturated fatty acids were detected, but were present at extremely low percentages. Majorsn-2 fatty acids in alkylacyl CPL were 16∶0 (25.4%), 22∶6n−3 (16.0%) and 20∶5n−3 (8.4%). The major fatty acids of diacyl EPL were 20∶5n−3 (22.3%), 16∶0 (17.9%), and 18∶0 (16.1%), and those of diacyl CPL were 16∶0 (30.4%), 20∶5n−3 (17.6%) and 18∶1n−7 (7.4%).     

Positional distribution on n−3 fatty acids in triacylglycerols from rat adipose tissue during fish oil feeding

The present study was designed to investigate the metabolism of the n−3 olyunsaturated fatty acids (PUFA) in adipose tissue and its dependence upon dietary factors. Changes in the positional distribution of the fatty acids in triacylglycerols from retroperitoneal adipose tissue were studied as a function of time on rats fed for 4 wk a diet enriched with fish oil. The stereospecific analysis of triacylglycerols was based on random formation ofrac-1,2-diacylglycerols by Grignard degradation. This was followed by synthesis ofrac-phosphatidic acids and treatment with phospholipase A₂. In the triacylglycerols of the fish oil diet, 57% of the total n−3 fatty acids were in position 3,i.e., two-thirds of 22∶5n−3 and 22∶5n−3 were esterified insn-3 position, whereas 22∶6n−3 was equally distributed in positions 2 and 3. After 4 wk of feeding fish oil, the fatty acid composition of adipose tissue triacylglycerols reached a steady state. Half of the n−3 fatty acids were found in position 3, namely 75% of 22∶5n−3, 50% of 20∶5n−3 and 18∶4n−3 and 45% of 22∶6n−3, the latter being equally distributed in positions 2 and 3. This pattern of distribution resembled that found in triacylglycerols of the fish oil diet, except for a higher proportion of 20∶5n−3 in adipose tissue in position 1 at the expense of position 3. Throughout the 4-wk period of fish oil feeding, the distribution pattern of minor n−3 fatty acids (18∶4n−3 and 22∶5n−3) in adipose tissue triacylglycerols remained unchanged. On the other hand, at the onset of fish oil feeding, 20∶5n−3 and 22∶6n−3 became concentrated in position 3, but thereafter 20∶5n−3 was progressively incorporated into position 1 and 22∶6n−3 into position 2. We thus conclude that n−3 fatty acids are differentially esterified in triacylglycerols of white adipose tissue. Despite the complex sequence of hydrolysis and acylation steps involved, the positional distribution of n−3 fatty acids was found to be similar in both the fish oil diet and the stored fat, in contrast to what was observed for nonessential fatty acids.     

The effect of lyophilization on the solvent extraction of lipid classes,fatty acids and sterols from the oysterCrassostrea gigas

The lipid class compositions of adult Pacific oysters [Crassostrea gigas (Thunberg)] were examined using latroscan thin-layer chromatography/flame-ionization detection (TLC/FID), and fatty acid compositions determined by capillary gas chromatography and gas chromatography/mass spectrometry (GC/MS). The fatty acid methyl esters were separated using argentation TLC and also analyzed as their 4,4-dimethyloxazoline derivatives using GC/MS. Major esterified fatty acids inC. gigas were 16∶0, 20∶5n−3, and 22∶6n−3. C₂₀ and C₂₂ nonmethylene interrupted (NMI) fatty acids comprised 4.5 to 5.9% of the total fatty acids. The NMI trienoic fatty acid 22∶3(7,13,16) was also identified. Very little difference was found in the proportions of the various lipid classes, fatty acids or sterols between samples of adult oysters of two different sizes. However, significant differences in some of the lipid components were evident according to the method of sample preparation used prior to lipid extraction with solvents. Lyophilization (freeze drying) of samples led to a significant reduction in the amounts of triacylglycerols (TG) extracted by solvents in two separate experiments (7.0 and 52.5% extracted). Extracts from lyophilized samples had less 16∶0, C₁₈ unsaturated fatty acids, and 24-ethylcholest-5-en-3β-ol, while C₂₀ and C₂₂ unsaturated fatty acids comprised a higher proportion of the total fatty acids. There was no significant change in the amounts of polar lipids, total sterols, free fatty acids or hydrocarbons observed in extracts from lyophilized samples relative to extracts from nonlyophilized samples. Addition of water to the freezedried samples prior to lipid extraction greatly improved lipid yields and resulted in most of the TG being extracted.     

Positional analysis of triglycerides and phospholipids rich in long-chain polyunsaturated fatty acids

Four sources of long-chain polyunsaturated fatty acids (LCP) differing in their chemical structure (triglycerides or phospholipids) and in their origin (tuna triglycerides, fungal triglycerides, egg phospholipids, and pig brain phospholipids) were analyzed to determine the distribution of the component fatty acids within the molecule. Lipase and phospholipase A₂ hydrolysis was performed to obtain 2-monoacylglycerols and lysophospholipids, respectively, which allowed us to determine the distribution of fatty acids between the sn-2 and sn-1,3 positions of triglycerides or between the sn-1 and sn-2 position of phospholipids. Fatty acids in the LCP sources analyzed were not randomly distributed. In tuna triglycerides, half of the total amount of 22∶6n−3 was located at the sn-2 position (49.52%). In fungal triglycerides, 16∶0 and 18∶0 were esterified to the sn-1,3 (92.22% and 91.91%, respectively) 18∶1 and 18∶2 to the sn-2 position (59.77% and 62.62%, respectively), and 45% of 20∶3n−6 and only 21.64% of 20∶4n−6 were found at the sn-2 position. In the lipid sources containing phospholipids, LCP were mainly esterified to the phosphatidylethanolamine fraction. In egg phospholipids, most of 20∶4n−6 (5.50%, sn-2 vs. 0.91%, sn-1) and 22∶6n−3 (2.89 vs. 0.28%) were located at the sn-2 position. In pig brain phospholipids, 22∶6n−3 was also esterified to the sn-2 (13.20 vs. 0.27%), whereas 20∶4n−6 was distributed between the two positions (12.35 vs. 5.86%). These results show a different fatty acid composition and distribution of dietary LCP sources, which may affect the absorption, distribution, and tissue uptake of LCP, and should be taken into account when supplementing infant formulas.     

Fatty acids and fatty alcohols of wax esters in the orange roughy: Specific textures of minor polyunsaturated and branched-chain components

Open-tubular gas chromatography was carried out on fatty acids and alcohols obtained from wax esters of the orange roughy,Hoplostethus atlanticus, caught at sea off New Zealand. The major (above 5%) components were 16∶1(n−7), 18∶1(n−9) and (n−7), 20∶1(n−9) and (n−7), and 22∶1(n−11, n−13) as fatty acids, and 16∶0, 18∶0, 18∶1(n−9), 20∶1(n−9) and (n−7), and 22∶1(n−11, n−13) as fatty alcohols. The total percentages of the minor components were 10% in the acids and 26% in the alcohols. The 22∶1/20∶1 ratio of the fatty alcohols obtained in this study was less than 1.0, although the ratio for the Atlantic orange roughy has been reported as being greater than 1.0. The contents of polyenes were as low as 2.48% in the acids and 0.95% in the alcohols, but their compositions showed some specific features. The percentages of the C₁₆−C₂₂ dienes in the total polyenes were remarkably high, 57.7% of these acids and 53.1% of these alcohols. The most important dienes were 18∶2(n−6) in the acids and 20∶2(n−6) in the alcohols.     

The steryl ester and phospholipid fatty acids of the spongeAgelas conifera from the Colombian Caribbean

The steryl ester and phospholipid fractions of the marine spongeAgelas conifera were isolated and analyzed. The fatty acyl components of the steryl ester and phospholipid fractions as determined by gas chromatography and gas chromatography/mass spectrometry were very similar and consisted of 56.8 and 62.7% of C₁₄−C₂₀ acids (normal; branched, especiallyiso andanteiso; and monounsaturated, particularly Δ⁹ and Δ¹¹ acids) and of 43.1 and 35.5% of C₂₄−C₂₆ acids (Δ^5,9 diunsaturated acids), respectively. The major constituent fatty acids detected were 13-methyltetradecanoic,n-hexadecanoic, 10-methylhexadecanoic, 11-octadecenoic, 12-methyloctadecanoic, 5,9-pentacosadienoic and 5,9-hexacosadienoic acids. The phospholipids isolated were identified as phosphatidylcholine (37%), phosphatidylserine (34%), phosphatidylethanolamine (16%) and phosphatidylinositol (11%). The distribution of fatty acids within the phospholipid classes was also determined.     

Fatty acids of unusual double-bond positions and chain lengths found in rat skin surface lipids

The fatty acids of rat skin surface lipids comprise four main skeletal types of chains which occur both as saturates and monoenes and range from C₁₂ to C₃₈: straight even, straight odd, iso and anteiso (the latter two identified by GC retention data only). Two unidentified series of branched monoenes also occur in trace amounts. Reductive ozonolysis of monoenes reveals two characteristic double-bond position patterns, one for the straight even chain series and the other for the straight odd chain series. The straight even chain pattern comprises four series, of which ω7 ≫ω9>ω5>ω11; the straight odd chain series in contrast shows a large number of ω series with irregular distribution. The biosynthesis of the even chain fatty acid monoenes can be thought of as occurring in two stages: synthesis of 14∶Δ9, 16∶Δ9, 18∶Δ9 and 20∶Δ9, with 16∶Δ9 predominating; elongation of these chains mostly by 1, 2, or 3 C₂ units but up to the unusually long lengths by 11 C₂ units. For the formation of the former, two schemes by known pathways are proposed. Iso and anteiso chains which are nearly all saturated comprised 1/3 the total fatty acids. Special terms and abbreviations: Normal even=a straight chain with an even number of carbon atoms, normal odd=a straight chain with an odd number of carbon atoms, ω=terminal carbon atom, iso=a straight chain with a methyl group at the ω−1 position, anteiso=a straight chain with a methyl group at the w−2 position, Δn=a double bond between the n^th and the (n+1)^th carbon atom from the carbonyl group of the fatty acid or ester, ωn=a double bond between the ω∩n^th and the ω-(n−1)^th carbon atom where n is an integer, aldester=aldehyde methyl ester, Me=methyl, GLC=gas-liquid chromatography, TLC=thin-layer chromatography.     

Specific distribution of fatty acids in the milk fat triglycerides of goat and sheep

The triglycerides of the fat globules of sheep and goat milk were isolated and separated into short and long chain lengths by silicic acid column chromatography. The short chain lengths comprised major triglycerides with 34–44 acyl carbon atoms and accounted for nearly 50% of the total milk fat. The long chain lengths contained major triglycerides with 40–54 acyl carbons. Stereospecific analyses of the short chain triglyceride fraction showed that of the 20–23 moles per cent of C₄−C₈ fatty acids present, at least 95% were specifically attached to the glycerol molecule in the position corresponding to carbon 3 ofsn-glycerol. The distribution of the other fatty acids (C₁₀ or greater) did not show such marked specificity for either the 1 or the 2 position. Although individual triglycerides were not identified, the specific placement of the fatty acids could best the accounted for by assuming a common pool of long chain 1,2-diglycerides which served as precursors of the bulk of both short and long chain triglycerides during milk fat synthesis. Presented in part at the AOCS Meeting, New York, October 1968.     

The fatty acid composition of a Vibrio alginolyticus associated with the alga Cladophora coelothrix. Identification of the novel 9-methyl-10-hexadecenoic acid

The fatty acid composition of a new strain of Vibrio alginolyticus, found in the alga Cladophora coelothrix, was studied. Among 38 different fatty acids, a new fatty acid, 9-methyl-10-hexadecenoic acid and the unusual 11-methyl-12-octadecenoic acid, were identified. Linear alkylbenzene fatty acids, such as 10-phenyldecanoic acid, 12-phenyldodecanoic acid and 14-phenyltetradecanoic acid, were also found in V. alginolyticus. The alga contained 43% saturated fatty acids, and 28% C₁₆–C₂₀ polyunsaturated fatty acids of the n−3 and n−6 families.     

Isolation and characterization of the monounsaturated long chain fatty acids ofMycobacterium tuberculosis

The positions of double bond in the monounsaturated C₁₅−C₃₂ fatty acids ofMycobacterium tuberculosis H37Ra were established by gas chromatography/mass spectrometry of the ozonized esters and their pyrrolidide derivatives. The monounsaturated C₁₅−C₂₁ fatty acids had the double bond primarily at the Δ⁹ position while the monounsaturated longer chain fatty acids (C₂₂−C₃₂) had the double bond in several positions. Many of the latter acids, especially the odd-numbered series, were very complex isomeric mixtures. Quantitation showed the most abundant even-numbered long chain fatty acid isomers to be as follow: C₂₂, Δ⁴; C₂₄, Δ⁵; C₂₆, Δ⁷ and Δ⁹; C₂₈, Δ⁹; C₃₀, Δ¹¹ and Δ¹³; C₃₂, Δ¹³ and Δ¹⁵.     

The content and composition of sterols and sterol esters in sunflower and poppy seed oils

The composition and proportion of free sterols and sterol esters in crude sunflower and poppy seed oils were determined, using preparative thin layer chromatography followed by gas chromatography with cholesterol as an internal standard. Free sterols and sterol esters were also isolated in a liquid fraction obtained by low temperature crystallization (−80 C) of the oils and enriched with minor lipid classes. This enrichment procedure provided a liquid fraction suitable for studies of minor components in the oils. However, selectivity towards sterol esters was observed since sterols esterified to very long chain fatty acids (C₂₀–C₂₄) were preferentially retained in the precipitate. The proportions of free and esterified sterols were found to be 0.34 and 0.28%, respectively, in the sunflower oil, whereas the corresponding figures for poppy seed oil were 0.33% and 0.05%. Sunflower oil was characterized by a relatively high percentage of Δ7-sterols, preferentially obtained in the esterified fraction, and by very long chain saturated fatty acids of sterol esters. The sterols in poppy seed oil were composed almost entirely of campesterol, stigmasterol, sitosterol and Δ5-avenasterol, although their percentage distributions were remarkably different in the free and esterified fraction.     

The effect of a fat free diet on esterified monoenoic fatty acid isomers in rat tissues

Rats were maintained 120–140 days on a normal diet (group 1) or one deficient in fatty acids (group 2). Isomer composition was determined of monoenoic fatty acids (16∶1, 18∶1) isolated from total lipids of heart, kidney, lung, brain and lumbar fat, and from separated neutral lipids and phospholipids of heart and kidney. Group 1: The number of major isomers of C_16∶1 and C_18∶1 was similar in all tissues but their proportions varied in different tissues and types of lipid. Group 2: The proportions of 16∶1(n−7) increased and of other 16∶1 isomers decreased in all tissues; 18∶1(n−9) was increased at the expense of (n−7) in heart, to a lesser extent in kidney, and was little changed in lung, lumbar fat, or brain. The decrease in proportion of 18∶1(n−7) was greatest in heart-muscle phospholipids. C_20∶3 comprised 95% (n−9) and 5% (n−7) in heart and kidney lipids. The changes in group 2 probably represent the body’s attempts to maintain lipids with the physical and chemical properties necessary to normal biological function. Nomenclature of fatty acids as in IUPAC-IUB Commission on Biochemical Nomenclature, “The nomenclature of lipids,” J. Biol. Chem. 242:4845–49 (1967).     

Neutral lipids of chickpea flour and protein isolates

The neutral lipids composition of defatted chickpea flour and two types of protein isolates has been studied. The main compounds in neutral lipids are triacylglycerols, free fatty acids, and diacylglycerols. Other compounds present are wax esters, free fatty alcohols, and free sterols. The main fatty acids in neutral lipids are C_18:2 and C_18:1 among the unsaturated, and C_16:0 and C_18:0 among the saturated acids. Free and esterified alcohols range from C_16:0 to C_28:0, the majority being those with an even number of carbon atoms. Sterols observed are β-sito-sterol, campesterol, stigmasterol, and δ-5-avenasterol. Triacyl-glycerols are partially hydrolyzed, and the amounts of unsaturated sterols and unsaturated fatty acids are reduced as a result of the chemical treatment during production of the protein isolates.