Akira Shibahara

Determination of double-bond positions in methylene-interrupted dienoic fatty acids by GC-MS as their dimethyl disulfide adducts

Abstract A method using GC-MS for determining the double-bond positions in methylene-interrupted dienoic fatty acids as their dimethyl disulfide adducts is described. As a standard procedure for the single-step preparation of the adducts, methyl linoleate was subjected to the I 2 -catalyzed reaction with dimethyl disulfide for 30 min. After the addition of a hexane/ether mixture and an aqueous Na 2 S 2 O 3 solution to reduce I 2 , the resulting adducts in the upper phase were analyzed directly by GC-MS. The adducts consisted of an equimolar mixture of methyl 9,10-bis(methylthio)octadec-12-enoate and 12,13-bis(methylthio)octadec-9-enoate. The cleavage between the methylthio-substituted carbons yielded sets of key fragment ions showing the original double-bond positions in the starting ester. Standard experiments using other authentic methyl dienoates revealed the accuracy of this convenient method. The analysis of [2,2- 2 H 2 ]linoleic acid and naturally-occurring dienoic fatty acids using this method is also described.

cis-Vaccenic acid in mango pulp lipids

A peak corresponding to a methyl octadecenoate other than oleate has been detected on the capillary gas chromatogram of the methyl esters of mango pulp fatty acids. This octadecenoate was isolated by silica gel and argentation column chromatography, high performance liquid chromatography and argentation thin layer chromatography, and then analyzed by infrared, nuclear magnetic resonance and gas chromatography-mass spectrometry, chromatographic separations and oxidative degradation. These analytical data proved that the octadecenoic acid wascis-vaccenic acid (cis-11-octadecenoic acid). The concentration of this acid in total octadecenoic acids ranged from 35% to 50% in the pulp of mangoes from Fiji, Mexico, the Philippines and Taiwan.cis-Vaccenic acid was revealed to be one of the major component fatty acids of non-polar lipids (mainly triacylglycerols), glycolipids and phospholipids in mango pulp. The glycolipids containedcis-vaccenic acid (ca. 20%) in higher concentration than oleic acid (ca. 15%). A trace amount ofcis-vaccenic acid (0.5%) was detected in the total lipids of mango seeds. Profile of fatty acid composition of mango pulp lipids (0.2–0.3 wt% of wet pulp) was characterized by the presence of n−7 acid isomers,cis-vaccenic acid and palmitoleic acid, and unusual mono- and dienoic positional isomers.

Novel pathways of oleic and cis-vaccenic acid biosynthesis by an enzymatic double-bond shifting reaction in higher plants

The novel pathways of oleic acid formation from cis‐vaccenic (cis‐11‐octadecenoic) acid and of cis‐vaccenic acid formation from oleic acid by enzymatic positional isomerization have been proposed in higher plants, based on stable‐isotope experiments using[2,2‐2H2]cis‐vaccenate or [2,2‐2H2]oleate as an immediate precursor. A pulp homogenate and also pulp slices prepared from developing kaki (Diospyros kaki) fruit could catalyze these hitherto unknown isomerizations. This suggests the presence of a new type of isomerase responsible for the double‐bond shifting reaction without cis‐trans isomerization in the middle of fatty acid carbon chains.

cis-Vaccenic acid in pulp lipids of commonly available fruits

Fatty acids of commonly available fruit pulps have been analyzed by capillary gas chromatography of their methyl esters and by gas chromatography-mass spectrometry as their dimethyl disulfide adducts. The gas chromatographic and mass spectrometric data proved that these fruits always containedcis-vaccenic (cis-11-octadecenoic) acid as a component fatty acid of their pulp lipids. The concentration ofcis-vaccenic acid in total octadecenoic acids ranged from 1.9% to 95.1% in the fruit pulps examined. The highest concentration of this acid was detected in pulp lipids of Japanese persimmon (Diospyros kaki). In fruit pulp lipids,cis-vaccenic acid was a common octadecenoic acid as well as oleic acid.

Application of a GC-MS method using deuterated fatty acids for tracingcis-vaccenic acid biosynthesis in kaki pulp

A gas chromatographic-mass spectrometric method using [2,2-2H2]fatty acids has been developed to trace the biosynthesis ofcis-vaccenic (cis-11-octadecenoic) acid in higher plants. The deuterated fatty acids and other unlabeled fatty acids in the biosynthetic reaction mixture were converted into bis(methylthio) derivatives and analyzed by mass chromatography. The principle of this method was based on the shift of key fragment ions (containing two deuterium atoms) due to the cleavage between the methylthio-substituted carbons. The labeled compounds were detected by the m/z values which shifted 2 mass units from those of the corresponding unlabeled compounds and estimated by a calibration curve based on the peak areas of the key fragment ions. For metabolic experiments, a homogenate fraction was prepared from the pulp part of maturing kaki (Diospyros kaki) fruit and incubated with ammonium [2,2-2H2]palmitoleate (cis-9-hexadecenoate) or [2,2-2H2]palmitoleoyl-CoA. The incubation resulted in the formation of detectable amounts of isotopically-labeledcis-vaccenic acid containing two deuterium atoms at the carbon chain between the double bond and the carboxyl group. This experimental evidence proved thatcis-vaccenic acid was formed from palmitoleic acid by chain elongation.

Double-bond localization in heneicosapentaenoic acid by a gas chromatography/mass spectrometry (GC/MS) method

Double-bond locations in heneicosapentaenoic acid from eel lipids were determined by a method involving gas chromatography/mass spectrometry (GC/MS). The methyl esters of the pentaenoate fraction were first partially reduced with hydrazine. The resultingcis-monoenoates were then methylthiolated, and the resultant adducts were analyzed by GC/MS. The key fragmentation ions generated by the cleavage between the methylthio-substituted carbons were used to ascertain the original double-bond positions in the native fatty acid esters. Based on mass spectral evidence, the acid was identified as all-cis-6,9,12,15,18-heneicosapentaenoic acid.

Occurrence of n-5 monounsaturated fatty acids in jujube pulp lipids.

The pulp lipids of jujube (Zizyphus jujuba var.inermis) fruit have been shown by chromatographic, spectrometric and chemical analyses to contain a series ofcis-monoenoic fatty acids with n−5 unsaturation as major acyl moieties. The total concentration of these n−5 fatty acids, such as 14∶1n−5, 16∶1n−5 and 18∶1n−5, ranged from 22 to 54% of total fatty acids in the pulp lipids of 11 different sources. The main component of the n−5 homologues was 16∶1n−5 in all cases. Other monoenoic acids with n−7 unsaturation, namely palmitoleic (cis-9-hexadecenoic) acid andcis-vaccenic (cis-11-octadecenoic) acid, as well as with n−9 unsaturation, namely oleic acid, were also identified. In the seed lipids of jujube fruit, none of the n−5 monoenoic acids could be detected. Thus the jujube pulp lipids are characterized by the predominance of n−5 monoenoic acid isomers.

Toxic Character of Rancid Oil. XVI.

In the previous papers, the authors reported that tocopherol showed the effect to reduce the toxicity of thermally oxidized oil (TO) in rats.The present paper deals with the comparison of nutritive values among TO with or without tocopherol (Toc) and fresh oil without tocopherol.Soybean oil was heated at 180°C for 50 hours, and TO with or without Toc and/or fresh oil (FO) from which Toc was removed by active alumina column chromatography were fed to rats in a diet contained 15% of test oils.The results indicated that the rats fed with TO showed growth depression as reported in the previous papers. On the other hand better growth was observed when the rats were fed with the Toc free FO.The weight and lipid content in liver and kidney of rats fed with TO were higher than those of rats fed FO or Toc free FO.On the contrary, the decrease of weight, lipid and Toc content in testicle of rats fed with TO or Toc free FO was observed.Feeding of TO and Toc free FO reduced the percentage of C18 : 2 in liver, kidney, testicle and blood lipids, but they were increased by the addition of Toc in the diets.Hemolysis rates of erythrocyte of rats fed with TO was higher than those of rats fed with free FO or the TO with Toc.

Decomposition of tocopherol, amino acids and ascorbic acid in ethanol solution by oxidized products of oil during autoxidation.

分別法を異にする方法で加熱大豆油から得た酸化酸 (OFA, 石油ェーテル不溶性の酸化脂肪酸) と脂肪酸重合物ならびにその二次酸化生成物 (SP, カラムクロマトグラフィーで分別) の性質および栄養成分の分解作用について比較した。1) OFAとSPのCOVは46.7および53.5, An. Vは562.0および640.6で, 両値ともSPのほうがわずかに高い。ペルオキシド, ェポキシド, アルデヒド, α-ケト酸の呈色反応はOFA, SPともに陽性であった。OFAおよびSPによりスーパーオキシドが発生した。2) TocとOFAおよびSPの1%添加混合物を60および20±2℃に保存した。60℃, 4日目でTocの残存率はOFAの場合32.0%, SPで27%であった。3) OFAの添加量が増すにしたがいアミノ酸は多く分解した。用いたアミノ酸中では, Cysが最も分解されやすく, ついで, His, Trpの順であった。SPとOFAとでは, SPよりもOFAのほうがアミノ酸の分解率がやや高い傾向にあった。4) OFAの添加量が増すにしたがいアスコルビン酸の分解率も高くなった。アスコルビン酸に対するSPの作用はOFAとほぼ同程度であった。

Tocopherol(Toc) and sterol composition and concentration in Mallotus japonicus seeds.

Mallotus japonicus seeds were separated to cotyledons and seed coat. The lipid content in the whole seed was 45.2%; the cotyledon lipid content was higher than the seed coat content. The main components of seed Toc were α-, γ- and δ-Toc. δ-Toc was found at a higher level in the seed -coat than in the cotyledon. In whole seed lipids the percentages of campe-, stigma- and sitosterols in demethylsterol were 31.1, 10.7 and 58.0, respectively. Clear differences were not found in sterol compositions between free sterol and sterol esters. The main components of monomethylsterol in whole seed lipids were cycloeucalenol (including gramisterol) and 24-ethyllophenol, and the main components of dimethylsterol were cycloartanol, β-amyrin, cycloartenol and 24-methylene cycloartanol.