Analysis, Metabolism And Physiological Roles Of Conjugated Linolenic Acid

It was reported that conjugated linolenic acid (CLNA) have a cytotoxic effect on cultured human tumor cells, inhibit carcinogenesis and alter the lipid metabolism in animals. This has led to increased interest in CLNA. This thesis focuses on the main isomers of CLNA, a-eleostearic acid (a-ESA, c9, t11, t13-CLNA) and punicic acid (9c, t11, cl3-CLNA), and consists of three parts:part one investigated the analysis of CLNA; part two investigated the metabolism of CLNA in animals and humans, and part three investigated physiological roles in animals and humans.Part one:Analysis of conjugated linolenic acidDifferent methods for methylating CLNA were compared. The results showed that precise methylation methods should be chosen according to the chemical forms of CLNA. For lipids with negligible content of free fatty acid (FFA). it can be methylated by 0.5 N NaOMe/methanol (55℃,20 min). For lipids with considerable content of FFA, it can be methylated by two-step method:First, the lipids were hydrolyzed into FFA by 0.3 N KOH/90% ethanol (37℃,2 h). Second, the FFA was methylated by 1 N H2SO4/methanol (50℃,10 min).The lipid classes and CLNA content in seed oils of Trichosanthes kirilowii (TK), three pomegranate and six bitter melon cultivars were investigated. The predominant lipid was triacylglycerol (TAG) in the analyzed samples ranging from 92.6 to 97.0%. In Trichosanthes kirilowii seed oil, the major fatty acids were 18:1 (22.6%),18:2n-6 (32.6%) and PA (32.6%), and the total CLNA was 36.9%. In pomegranate cultivars seed oil, punicic acid was the most predominant fatty acid, ranging from 73.4 to 77.5%, and the total CLNA ranged from 83.4 to 87.9%. In bitter melon seed oils, the total CLNA ranged from 65.2 to 71.9%. and a-ESA was the predominant one, accounting for 56.0 to 63.7%. TK, pomegranate and bitter melon were rich in CLNA. However, the main CLNA in TK and pomegranate was PA, whereas in bitter melon it was a-ESA.The TK seed oil was extracted with two solvents, chloroform-methanol (C:M extract) and petroleum ether (PE extract). The oil content of TK seed was 33.3-34.6%. The predominant lipid component was TAG (95.8-98.4%) with smaller amounts of sterol ester, FFA, diacylglycerol, sterol and PL. The major fatty acids were linoleic acid (32.7-32.7%) and PA (32.6%-33.3%) followed by oleic acid (22.6-22.9%) in the total lipids (TL). The TAG fatty acid profile is very similar to TL, whereas there exit differences between PL and TL in fatty acid profile and C:M extract and PE extract in fatty acid profile of PL fraction. The PL fraction (2.4-9.4%) contained much less amounts of PA than TL (32.6%-33.3%).Part two:metabolism of conjugated linolenic acid in animals and humansThe incorporation and metabolism of PA in rat tissues and plasma, one isomer of CLNA was studied by oral administration of TK seed oil, a unique PA-containing material, to rats and which were monitored for 24 h. The metabolite was identified and analyzed by HPLC and GC-MS. The results show that PA was incorporated and metabolized to c9,t11-CLA in rat plasma, liver, kidney, heart, brain and adipose tissue. The level of PA and CLA in liver and plasma was higher than in brain, heart, kidney and adipose tissue, and the lowest accumulation occurred in the brain.The conversion rate into c9,t11-CLA of a-ESA and PA was investigated in the second part of this thesis. Six-week-old male ICR mice (n=8) were fed either a control diet or diets supplemented with 1%α-ESA or PA mixtures in the form of TAG for 6 wk. The results showed thatα-ESA and PA converted into the same metabolite, c9,t11-CLA in mice. The relative conversion rate in all mice tissues in theα-ESA group was higher than in the PA group. There were significant tissue differences in relative conversion rate in both a-ESA and PA groups. The highest conversion rate in the a-ESA group was found in the adipose tissue, kidney and spleen, and the lowest was in heart. However the highest conversion rate in the PA group was the liver and the lowest was in the heart. It is possible that geometrical structure of△13-double bond affected the△13-double saturation reaction activity of enzyme and the difference in conversion rates of a-ESA and PA in vivo was due to substrate-specific effect of this enzyme.The metabolism of PA was investigated in healthy young human subjects. The study was a randomized, placebo-controlled parallel trial. Thirty subjects received TK seeds containing 3g PA in the form of TAG or control for 4 wks. The results showed that PA can be incorporated into the human plasma and red cell membrane. Partial of PA can also be metabolized into c9, t11-CLA. The conversion rate of PA into CLA was 34% in human plasma whereas 28% in red cell membrane. To our knowledge, this is the first study demonstrating the incorporation and metabolism of CLNA in humans. It suggests that the△l3-double bond saturation reaction by which CLNA converted into c9,t11-CLA can also be occurred in humans as in rats and mice. However, further studies are warranted to provide a clearer understanding of this mechanism of CLNAPart three:physiological roles of conjugated linolenic acid in animals and humansThe effects of CLA, a-linolenic acid (ALA), a-ESA and PA on body fat, plasma lipids and liver lipids were investigated in mice. Six-week-old male ICR mice (n=8) were fed either a control diet or one of four experimental diets supplemented with 1% ALA,1% a-ESA,1% PA and 1% CLA mixture, respectively, in the form of TAG for 6 wks. Body weight and adipose tissue was significantly reduced by CLA feeding, whereas those were not modified by ALA, a-ESA or PA. There was no significant effect of CLA, ALA, a-ESA and PA on plasma lipids. However, the TAG content in the liver was significantly reduced by a-ESA and PA feeding. It suggests that there exits differences in the physiological roles among CLNA and CLA although the CLNA can be metabolized into CLA in vivo.The influence of a-ESA and PA on fatty acid profiles in liver, kidney, heart, spleen and adipose tissue was investigated in mice, comparing with CLA. Six-week-old male ICR mice (n=8) were fed either a control diet or diets supplemented with 1% a-ESA, PA or CLA mixtures in the form of TAG for 6 wks. The content of 18:2n-6 was significantly reduced in heart and adipose tissue, correspondingly that of total n-6 PUFA and PUFA were significantly reduced in adipose tissue by CLA and CLNA feeding. The content of 18:2n-6 in kidney was also reduced by PA feeding. The content of 22:6n-3 was significantly increased in liver, kidney and heart by PA feeding, leading the increasing total n-3 PUFA content whereas those were not modified by a-ESA feeding in all tissues examined. In contrast to PA. supplementation of CLA significantly decreased the 22:6n-3 content in liver, heart. Feeding CLA also decreased the level of 20:4n-6 in liver, kidney and heart, whereas CLNA have no effect on that of 20:4n-6 in all tissues examined. CLNA incorporation and conversion to CLA could also exert beneficial activities in different tissues through the effect of the metabolite CLA or CLNA solely.The effect of PA on plasma lipids. inflammatary parameters and lipid peroxidation was investigated in healthy young humans. The study was a randomized, placebo-controlled parallel trial. Thirty subjects received TK seeds containing 3g PA in the form of TAG or control for 4 wks. The results showed that plamsa lipids and inflammatary parameters were not significantly affected, whereas the level of 8-iso-prostaglandinF2a in the urine, a lipid oxidation indicator in vivo was significantly elevated by PA supplemenation. Further studies of the mechanism behind, and the possible consequences of, the increased lipid peroxidation after PA supplementation are needed.