Diversity in γ-oryzanol profiles of Japanese black-purple rice varieties.

The γ-oryzanol contents and the composition of steryl ferulates distributed in Japanese pigmented rice varieties were investigated using the high-performance liquid chromatography-ultraviolet detection method for the purpose of expanding their utilisation. The average γ-oryzanol content in nine black-purple, four red, four green and three brown rice varieties was 54.2, 47.3, 44.3 and 43.3 mg γ-oryzanol equivalent/100 g dried weight, respectively. Among the nine varieties of black-purple rice, five varieties showed steryl ferulate composition similar to that of brown, red and green varieties. In contrast, the composition of steryl ferulates in other four black-purple rice varieties was partially specific and was characterised by a low amount of campesteryl ferulate and high of campestanyl ferulate and stigmastanyl ferulate. The latter two steryl ferulates have been recognised as minor components of γ-oryzanol in rice and as major components in wheat and corn. These results indicate that the compositions of steryl ferulates vary among Japanese black-purple rice varieties.

Simultaneous Synthesis of Vitamins D2, D4, D5, D6, and D7 from Commercially Available Phytosterol, ß-Sitosterol, and Identification of Each Vitamin D by HSQC NMR.

We succeeded in simultaneously synthesizing the vitamin D family, vitamins D2, D4, D5, D6, and D7, from ß-sitosterol, which is sold as a commercially available reagent from Tokyo Chemical Industry Co., Ltd. It is officially sold as a mixture of four phytosterols {ß-sitosterol (40-45%), campesterol (20-30%), stigmasterol, and brassicasterol}. Owing to this, we anticipated that, using this reagent, various vitamin D analogs could be synthesized simultaneously. We also synthesized vitamin D3 from pure cholesterol and analyzed and compared all vitamin D analogs (D2, D3, D4, D5, D6, and D7) by HSQC NMR. We succeeded in clearly demonstrating the difference in the NMR chemical shifts for each vitamin D analog.

Synthesis of three deoxy-sophorose derivatives for evaluating the requirement of hydroxy groups at position 3 and/or 3' of sophorose by 1,2-ß-oligoglucan phosphorylases.

Sophorose (Sop2) is known as a powerful inducer of cellulases in Trichoderma reesei, and in recent years 1,2-ß-D-oligoglucan phosphorylase (SOGP) has been found to use Sop2 in synthetic reactions. From the structure of the complex of SOGP with Sop2, it was predicted that both the 3-hydroxy group at the reducing end glucose moiety of Sop2 and the 3'-hydroxy group at the non-reducing end glucose moiety of Sop2 were important for substrate recognition. In this study, three kinds of 3- and/or 3'-deoxy-Sop2 derivatives were synthesized to evaluate this mechanism. The deoxygenation of the 3-hydroxy group of D-glucopyranose derivative was performed by radical reduction using a toluoyl group as a leaving group. The utilization of a toluoyl group that plays two roles (a leaving group for the deoxygenation and a protecting group for a hydroxy group) resulted in efficient syntheses of the three target compounds. The NMR spectra of the two final compounds (3-deoxy- and 3,3'-dideoxy-Sop2) suggested that the glucose moiety of the reducing end of Sop2 can easily take on a furanose structure (five-membered ring structure) by deoxygenation of the 3-hydroxy group of Sop2. In addition, the ratio of the five- and six-membered ring structures changed depending on the temperature. The SOGPs exhibited remarkably lower specific activity for 3'-deoxy- and 3,3'-dideoxy-Sop2, indicating that the 3'-hydroxy group of Sop2 is important for substrate recognition by SOGPs.

Degradation of Fucoxanthin to Elucidate the Relationship between the Fucoxanthin Molecular Structure and Its Antiproliferative Effect on Caco-2 Cells.

Fucoxanthin has an antiproliferative effect on cancer cells, but its detailed structure⁻activity correlation has not yet been elucidated. To elucidate this correlation, fucoxanthin was degraded by ozonolysis. The degraded compounds of fucoxanthin obtained by ozonolysis were purified by HPLC and analyzed by NMR. The polyene chain of fucoxanthin was cleaved by ozonolysis, and the fucoxanthin was divided into two types of cyclohexyl derivatives, one with a ß,γ-epoxy ketone group and the other with an allenic bond. In order to elucidate the structure⁻activity correlation, Caco-2 cells (human colorectal carcinoma) were treated with fucoxanthin degradation compounds. It was found that the entire structure of fucoxanthin is not essential for its antiproliferative effect and that even a partial structure exerts this effect.

The content and distribution of steryl ferulates in wheat produced in Japan.

Oryzanol contained in rice bran is a complex mixture of steryl ferulates (SFs) with many identified health benefits. Recently, SF has been shown to exist in other cereals such as wheat, rye, and corn. In this study, SFs in several wheats produced in Japan were analyzed. For instance, SF content of whole wheat grain, Yumekaori (Japan) was 15.2 ± 1.4 mg-oryzanol-equivalent/100 g grain, while that of the imported one, 1CW (Canada) was 11.4 ± 1.3 mg-oryzanol-equivalent/100 g grain. The main SF components in the examined wheats were campesteryl ferulate, campestanyl ferulate, and sitostanyl ferulate. SF distribution in whole wheat grain was investigated using 14 fractions produced by a conventional test milling machine. SF was intensively accumulated in the four bran fractions (24 - 95 mg-oryzanol-equivalent/100 g bran fraction). These results suggest that the wheat bran would be an important source of SF.

Inconsistencies in a highly polar capillary gas chromatography column and necessity of column performance checks for trans fatty acid measurement.

GC with a capillary column (60-100 m length) is widely used to measure trans fatty acids in dietary fats and biological tissues. Recently, we have occasionally observed that isothermal operation of an SP-2560 column at 180 degrees C results in incomplete separation of gondoic acid (11c-20:1) and one of the geometric isomers of alpha-linolenic acid (9t,12c,15c-18:3), although it has been known to produce their baseline separation in American Oil Chemists' Society Official Method Ce 1h-05, as well as in previous studies. Thus, trans isomer (9t,12c,15c-18:3) is one of the main components of trans fatty acids in refined edible oils, and the baseline separation of this peak from that of 11c-20:1 is indispensable. We demonstrate in this study that an isothermal operating temperature of 175 degrees C for an SP-2560 column results in satisfactory resolution of these two fatty acids. Because of the inconsistency in the separation provided by SP-2560 columns, careful monitoring of the relative elution order of different fatty acid methyl esters using standards is necessary for the exact evaluation of trans fatty acid contents in oils and fats.

cis-trans isomerization of carbon double bonds in monounsaturated triacylglycerols via generation of free radicals.

We investigated the heat-induced cis/trans isomerization of double bonds in monounsaturated lipids. When triolein (9-cis, 18:1) was heated around 180 degrees C, small amounts of isomerization products were obtained depending on the heating period. The heat-induced isomerization of triolein was considerably suppressed by the addition of different antioxidants or under nitrogen stream, and these additives simultaneously inhibited the thermal oxidation of double bonds in triolein. Therefore, an intermediate of the thermal oxidation reaction might be responsible for the heat-induced isomerization of the double bonds in triolein. The thermodynamics of the heat-induced isomerization of triolein (9-cis, 18:1) and trielaidin (9-trans, 18:1) were investigated using Arrhenius plot. The Arrhenius activation energies of cis double bonds in triolein and trans double bonds in trielaidin were 106 kJ/mol and 137 kJ/mol, respectively. The calculated internal rotational barrier heights of these double bonds were similar to those of the double bond of 2-butene radical and significantly lower than those of non-radicalized double bonds in 2-butene. These results suggest that heat-induced cis/trans isomerization of triolein and trielaidin occurs mainly through the formation of radical species, which are the intermediates produced during thermal oxidation. The activation energy difference between the two forms suggests that trans trielaidin radicals are more stable than cis triolein radicals. The high thermodynamic stability of the trans double bonds in lipid radicals would influence the population of cis and trans isomers in edible oils and contribute to slight accumulation of trans-18:1 isomers during heating or industrial processing.

Preparative separation of cis- and trans-isomers of unsaturated fatty acid methyl esters contained in edible oils by reversed-phase high-performance liquid chromatography.

In order to measure exactly the trans-fatty acids content in food materials, a preparative group separation of cis- and trans-isomers of unsaturated fatty acid methyl esters (FAMEs) was achieved by an isocratic reversed-phase HPLC (RP-HPLC) method. The trans-isomers of 16:1, 18:1, 18:2, 18:3, 20:1 and 22:1 FAMEs were readily separated from the corresponding cis-isomers by a COSMOSIL Cholester C18 column (4.6 mm I.D. x 250 mm, Nacalai Tesque) or a TSKgel ODS-100Z column (4.6 mm I.D. x 250 mm, TOSOH), using acetonitrile as the mobile phase. This method was applied for determining the trans-18:1 fatty acid content in partially hydrogenated rapeseed oil. The methyl esters of cis- and trans-18:1 isomers of the oil were collected as two separate fractions by the developed RP-HPLC method. Each fraction was analyzed by gas chromatography (GC) for both qualitative and quantitative information on its positional isomers. By a combination of RP-HPLC and GC methods, a nearly complete separation of cis- and trans-18:1 positional isomers was achieved and the trans-18:1 fatty acid content was able to be evaluated more precisely than is possible by the direct GC method. The reproducibility of cis- and trans-18:1 isomers fractionated by the RP-HPLC method was better than 98%. These results suggested that the preparative RP-HPLC method developed in this study could be a powerful tool for trans-fatty acid analysis in edible oils and food products as an alternative to silver-ion chromatography.

Absorption properties of micellar lipid metabolites into Caco2 cells.

To elucidate the absorption characteristics of dietary lipids in the human intestine, we investigated the cellular uptake of lipid metabolites using a differential monolayer of the Caco2 cells. As lipid metabolites, several free fatty acids and 2-monoacylglycerols, were formed a mixed micelle by bile salts and lysophospholipids and they were supplied to the Caco2 cells. To estimate the effect of the mixed micelles on the permeability of cells' membranes during incubation with the mixed micelles, the transepitherial electrical resistance (TEER) value was monitored, and no pronounced changes of TEER was detected. This suggested that mixed micelles did not affect their cellular properties of the barrier measured by TEER. The lipid metabolites transferred from the mixed micelle into the Caco2 cells were determined quantitatively by an enzymatic colorimetric method and were done by thin layer chromatography (TLC) for a species of acylglycerols. These highly sensitive methods enabled us to monitor the transepithelial transports of various kinds of non-isotope-labeled various lipid metabolites. Newly re-synthesized triacylglycerols were accumulated in Caco2 cells after 30 min incubation with the mixed micelles, and their amounts increased gradually for 4 h. The secretion of re-esterified triacylglycerols into a basolateral medium from the Caco2 cells began at 2 h after the mixed micelles were added to the apical medium. The intake of external lipid metabolites by the Caco2 cells were evaluated by an initial 2-h incubation with the mixed micelles. For example, 2-monomyristin and 2-monopalmitin were more rapidly transferred into the Caco2 cells from the mixed micelles than 2-monocaprin was. On the other hand, the absorption rates of capric acid, lauric acid and myristic acid by the cells were larger than those of stearic acid and oleic acid. It revealed that the side-chain structure of these lipid metabolites affected their absorption by the Caco2 cells. The results of this study suggested that the Caco2 cell monolayer could be a useful model for investigating the involvement of dietary lipids in the transepithelial absorption in the human intestine.

Intestinal epithelial cells absorb gamma-tocotrienol faster than alpha-tocopherol.

To elucidate the transepithelial transport characteristics of lipophilic compounds, the cellular uptake of tocopherol and tocotrienol isomers were investigated in Caco2 cell monolayer models. These vitamin E isomers formed mixed micelles consisting of bile salts, lysophospholipids, free fatty acid, and 2-monoacylglycerols, then the micelles were supplied to Caco2 cells. The initial accumulation of tocotrienol isomers in Caco2 cells was larger than those of corresponding tocopherol isomers. There was little difference among the cellular accumulations of four tocopherol isomers. These findings suggested that the difference between the molecular structures of the C16 hydrocarbon chain tail in tocopherol and tocotrienol was strongly responsible for the rapid epithelial transport into the Caco2 cells membranes rather than the difference in the molecular structures of their chromanol head groups. Furthermore, the secretion of alpha-tocopherol and gamma-tocotrienol from Caco2 cells was investigated using Caco2 cells plated on a transwell. The time courses of their secretions from Caco2 cells showed that the initial secretion rate of gamma-tocotrienol was also larger than that of alpha-tocopherol. To investigate the intestinal uptake of alpha-tocopherol and gamma-tocotrienol in vivo, the mice were fed single doses of alpha-tocopherol or gamma-tocotrienol with triolein. The gamma-tocotrienol responded faster in plasma than alpha-tocopherol, although the maximal level of gamma-tocotrienol was lower than that of alpha-tocopherol. This suggested that the intestinal uptake properties of administered alpha-tocopherol and gamma-tocotrienol would characterize their plasma level transitions in mice.

Acyl moieties modulate the effects of phospholipids on beta-carotene uptake by Caco-2 cells.

The intestinal absorption of carotenoids is thought to be mediated by the carotenoid assembly in mixed micelles, followed by its transfer into the enterocytes and subsequent secretion to the lymph as chylomicron particles. In the present study we investigated the effects of phospholipids and lysophospholipids with diverse fatty acyl moieties on the uptake of beta-carotene solubilized in mixed micelles by Caco-2 cells. Compared with phospholipid-free mixed micelles (NoPL), those containing long-chain PC inhibited beta-carotene uptake (16:0,18:1-PC approximately equal to 16:0,18:2-PC < 14:0,14:0-PC approximately equal to 16:0, 14:0-PC < 16:0,16:0-PC < NoPL). However, mixed micelles containing medium-chain PC enhanced beta-carotene uptake (NoPL < 8:0,8:0-PC < 12:0,12:0-PC < 10:0,10:0-PC), and short-chain PC did not affect the uptake. Among the lysophosphatidylcholine (LysoPC) class, a marked increase of beta-carotene uptake by medium-to-long-chain LysoPC was observed (NoPL < 12:0-LysoPC < 14:0-LysoPC < 18:1-LysoPC < 16:0-LysoPC), although short-to-medium-chain LysoPC (6:0-LysoPC to 10:0-LysoPC) did not affect beta-carotene uptake. The long-chain 16:0,18:1-PC increased the beta-carotene efflux from cells and drastically changed the beta-carotene UV-visible absorbance spectrum, compared with those of NoPL micelles. The acyl moieties of long-chain PC may interact with the carotenoid in the micelle interior, shifting the beta-carotene partition toward the micellar phase. Medium-chain PC and long-chain LysoPC, which have nearly equivalent hydrophobicities, may enhance beta-carotene uptake through their interaction with the cell membrane.

Acidolysis between triolein and short-chain fatty acid by lipase in organic solvents.

Ten kinds of lipases were examined as biocatalysts for the incorporation of short-chain fatty acids (acetic, propionic, and butyric acids) into triolein in order to produce one kind of reduced-calorie structured lipids. Trans-esterification (acidolysis) was successfully done in n-hexane by several microbial lipases. Among them, lipase from Aspergillus oryzae was used to investigate the effects of incubation time, substrate molar ratio, and water content on acidolysis. Finally, more than 80% of triolein was incorporated by butyric acid (molar ratio of triolein to butyric acid, 1:10) in the dried n-hexane at 52 degrees C for 72 h. More than 90% of the products was monosubstituent, which was esterified with this short chain fatty acid at the 1-position of the glycerol moiety of triolein. These results suggest that A. oryzae lipase would be a powerful biocatalyst for the synthesis of low caloric oil, such as triacylglycerol containing a mixture of long- and short-chain aliphatic acids.

Inhibitory effect of lysophosphatidylcholine on pancreatic lipase-mediated hydrolysis in lipid emulsion.

In the lipid metabolism pathway, dietary lipid emulsified with bile salts and phospholipids is mainly digested by pancreatic lipase into free fatty acids and monoacylglycerols. In order to study substrate recognition mechanism of a pancreatic lipase, we investigated its catalytic property toward the lipid emulsion prepared with long- or intermediate-chain acylglycerols and several physiological surfactants. When lysophosphatidylcholine (LysoPC), rather than bile salts or phospholipid, was incorporated into the lipid emulsion, it caused an increase in the Km(app) and a decrease in the Vmax(app) values in the interactions between the lipase and triacylglycerol (triolein or tricaprin). This indicated that LysoPC inhibited hydrolysis by decreasing both the substrate affinities and the catalytic activity of this lipase. Interestingly, further addition of taurodeoxycholic acid sodium salts or phospholipid completely restored the inhibitory effect of LysoPC on hydrolysis by lipase. On the other hand, the change in these kinetic values between the lipase and two 1-monoacylglycerols (1-monocaprin and 1-monoolein) were not particularly large when LysoPC was added. Particle size analysis of the lipid emulsion composed of LysoPC and triacylglycerols showed that most of the particles were less than 200 nm in size, which was smaller than the particle size in the triacylglycerol emulsions containing bile salts or phospholipid. The composition of the emulsion would affect its surface characteristics and thus contribute to changing lipase activity.

Polar organic solvent added to an aqueous solution changes hydrolytic property of lipase.

For developing further uses of lipase as a biocatalyst, its hydrolytic activity toward some esters was investigated in a miscible solution composed of a buffer and a polar organic solvent. Twenty percent dimethylformamide, 35% dimethylsulfoxide, 15% 1,4-dioxane, 15% dimethoxyethane, and 2% diethoxyethane promoted the hydrolysis by a lipase from Rhizomucor miehei toward some hydrophobic substrates, 4-methylumbelliferyl oleate, 4-methylumbelliferyl palmitate, and monoolein. While hydrolysis by this lipase toward the substrates with a relatively weak hydrophobicity (4-metylumbelliferyl heptanoate and 4-methylumbelliferyl nanoate) was suppressed by these solvents. A fluorometric analysis showed that the polar organic solvent in the buffer induced some conformational change around a tryptophan residue of R. miehei lipase. In addition to the influence of the miscible solvent on the solubility of the substrates, the conformational change of the protein induced by the miscible solvent would also affect the reactive properties of the lipase. Adding a polar organic solvent to an aqueous solution will be an efficient method for changing hydrolytic performance of lipases.

Fluorimetric analysis of lipase hydrolysis of intermediate- and long-chain glycerides.

For the purpose of deducing the digestive behavior of dietary fat in the digestive organs, a fluorimetric method for the measurement of hydrolysis by porcine pancreatic lipase was performed using intermediate- and long- acyl chain glycerides as substrates. Insoluble glycerides constituted by C10-C16 acyl chains were mechanically dispersed in 100% buffer and hydrolyzed by porcine pancreatic lipase. After the reaction, fatty acid released by the enzyme was extracted and its carboxyl group was fluorescently labeled with 9-bromomethylacridine. The 9-acridinylmethyl derivative of the fatty acid was separated and determined by HPLC. The sensitivity of this method was about 1000 times higher than that of the titrimetric method. Only 0.5 ng of porcine pancreatic lipase was sufficient for one routine assay. This assay method was successfully applied to investigate the enzymatic properties of porcine pancreatic lipase with respect to dietary lipids. The effects of some physiological factors concerned with lipid digestion, such as bile salt and colipase, on the lipase hydrolysis were also examined. The method established in the present study could contribute to a highly sensitive assay of some hydrolases containing lipase with regard to insoluble substrates.

Brown algae fucoxanthin is hydrolyzed to fucoxanthinol during absorption by Caco-2 human intestinal cells and mice.

The metabolic fate in mammals of dietary fucoxanthin, a major carotenoid in brown algae, is not known. We investigated the absorption and metabolism of fucoxanthin in differentiated Caco-2 human intestinal cells, a useful model for studying the absorption of dietary compounds by intestinal cells. Fucoxanthin was taken up by Caco-2 cells incubated with micellar fucoxanthin composed of 1 micromol/L fucoxanthin, 2 mmol/L sodium taurocholate, 100 micromol/L monoacylglycerol, 33.3 micromol/L fatty acids and 50 micromol/L lysophosphatidylcholine. Fucoxanthinol, the deacetylated product of fucoxanthin, was also found in both medium and cells, with its level increasing significantly in a time-dependent manner. No conjugated forms of fucoxanthin and fucoxanthinol were found in either medium or cells. In the animal study, fucoxanthinol (10.4 +/- 5.3 nmol/L plasma, n = 4) was detected in plasma of mice 1 h after intubation of 40 nmol fucoxanthin. These results indicate that dietary fucoxanthin is incorporated as fucoxanthinol, the deacetylated form, from the digestive tract into the blood circulation system in mammals.