Isotope ratio by HRGC-MS of citrus Junos tanaka (yuzu) essential oils: m/z 137/136 of terpene hydrocarbons.

The isotope ratios of monoterpene hydrocarbons in Citrus junos Tanaka (yuzu) essential oils from different origins were determined by ordinary high-resolution gas chromatography-mass spectrometry (HRGC-MS). Both intensities of the molecular mass peaks (m/z 136) and of the isotope peaks (m/z 137) of monoterpene hydrocarbons were measured by single-ion monitoring with an MS analysis. The isotope ratios (m/z 137/136) of the ten monoterpene hydrocarbons commonly contained in citrus essential oils, alpha-pinene, beta-pinene, sabinene, myrcene, alpha-phellandrene, alpha-terpinene, limonene, gamma-terpinene, beta-phellandrene and terpinolene, were determined in yuzu samples of the highest commercial quality from 42 different production districts. Statistical treatment of these data by the t-test and sign test revealed significant differences of the isotope effects in each yuzu sample. It is suggested that this technique will be applicable for evaluating the quality, genuineness and origin of citrus fruits and their products. The isotope fingerprints were also demonstrated in several citrus fruits other than the yuzu samples.

Radical-scavenging activities of citrus essential oils and their components: detection using 1,1-diphenyl-2-picrylhydrazyl.

Thirty-four kinds of citrus essential oils and their components were investigated for radical-scavenging activities by the HPLC method using 1,1-diphenyl-2-picrylhydrazyl (DPPH). To examine the oils' relative radical-scavenging activities compared with that of a standard antioxidant, Trolox was employed. All of the essential oils were found to have scavenging effects on DPPH in the range of 17. 7-64.0%. The radical-scavenging activities of 31 kinds of citrus essential oils were comparable with or stronger than that of Trolox (p < 0.05). The oils of Ichang lemon (64.0%, 172.2 mg of Trolox equiv/mL), Tahiti lime (63.2%, 170.2 mg of Trolox equiv/mL), and Eureka lemon (61.8%, 166.2 mg of Trolox equiv/mL) were stronger radical scavengers than other citrus oils. Citrus volatile components such as geraniol (87.7%, 235.9 mg of Trolox equiv/mL), terpinolene (87.4%, 235.2 mg of Trolox equiv/mL), and gamma-terpinene (84.7%, 227.9 mg of Trolox equiv/mL) showed marked scavenging activities on DPPH (p < 0.05).

Relationship between the reduction of tetrazolium salt XTT and DNA strand breakage with aminosugars.

Dihydropyrazine derivatives formed by the self-condensation reaction of D-glucosamine have the DNA breaking activity. To establish the monitoring method of the biological active dihydropyrazines, we investigated the relationship between the XTT (3'-[1-[(phenylamino)-carbonyl]-3, 4-tetrazolium]bis(4-methoxy-6-nitro)benzensulfonic acid hydrate) reducibility and the DNA breaking activity of aminosugars. Aminosugar in 50 mM sodium phosphate buffer (pH 7.4) was incubated at 37 degrees C. At a given time, the XTT reducibility and the DNA breaking activity of the incubated aminosugar were measured. Both XTT reducibility and DNA breaking activity showed a maximum value within 1-4 h after the incubation and then gradually decreased with the incubation time. Superoxide anion was suggested to involve in both of the DNA breaking activity and the XTT reducibility by the addition of the radical scavengers into those assay mixtures. The quantity of remaining covalently closed circular DNA and the XTT reducibility of all aminosugars showed a good correlation (r = 0.825, n = 26). This means that the XTT assay is applicable for the monitoring of those biologically active products derived from aminosugars when the participation of superoxide anion in DNA scission is recognized.

Isolation and characterization of free radical scavenging activities peptides derived from casein.

A peptide having the strong free radical scavenging activities was separated from casein protein hydrolysate by chromatographic analyses such as ion-exchange and gel filtration. SP-II fraction obtained by SP-Sephadex C-25 chromatography showed the most potent superoxide anion scavenging activity (SOSA), and it was further separated into a peptide using an octadecylsilano-high performance liquid chromatography. The amino acid sequence of the peptide was Tyr-Phe-Tyr-Pro-Glu-Leu (YFYPEL). The concentration of the test compound required to reduce the produced superoxide anion to one-half (IC(50)) value for SOSA was 79.2 microM using tetrazolium salt 3'-{1-[(phenylamino)-carbonyl]-3,4-tetrazolium}-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate method. The IC50 value for the 1,1-diphenyl-2-picrylhydrazyl radical and hydroxyl radical scavenging activities were 98 and 251 microM, respectively, based on the electron spin resonance method. We characterized SOSA of the C-terminal sequence using EL, PEL, YPEL, and FYPEL. The activities preferred sequences were EL>YFYPEL>FYPEL>YPEL>PEL, suggesting that the Glu-Leu sequence is important for the activity.

Reduction of tetrazolium salt XTT by aminoreductone formed during the Maillard reaction of lactose.

Lactose, a reducing disaccharide abundant in milk, reacts extensively with the amino groups of protein through the Maillard reaction. The Maillard reaction products showed 3'-[1-[(phenylamino)carbonyl]-3,4-tetrazolium]bis(4-methoxy-6-nitro)benzensulfonic acid hydrate (XTT) reducibility. The objective of this study was to clarify the Maillard reaction products responsible for the XTT reducibility. When lactose and butylamine were heated at 100 degrees C, the characteristic UV maximum at 320 nm was recognized and the relationship between the XTT reducibility and the compound with absorption maximum at 320 nm was investigated. The time course and the dependence on the heating temperature of the formation of the compound with absorption maximum at 320 nm were similar to those of the XTT reducibility. Their relationship showed a significant correlation (r = 0.967, n = 19). Furthermore, the spectrum change of the heated model solution by the addition of XTT suggested that the compound with absorption maximum at 320 nm would be involved in the reduction of XTT. Because the compound with absorption maximum at 320 nm was identified as an aminoreductone, 1-(butylamino)-1,2-dehydro-1,4-dideoxy-3-hexulose, by NMR analysis, it can be concluded that this was the main XTT-reducing substance.

Inhibitory effects of citrus essential oils and their components on the formation of N-nitrosodimethylamine.

Twenty-eight kinds of citrus essential oils and their components were studied for inhibitory effects on the formation of N-nitrosodimethylamine (NDMA). The reaction mixture consisted of dimethylamine and sodium nitrite adjusted at pH 3.6, in addition to essential oils and an emulsifying agent. The quantification was determined by high-performance liquid chromatography monitored at 220 nm. All of the essential oils inhibited the formation of NDMA in the range of 20-85%. The oils of ujukitsu (Citrus ujukitsu Hort. ex Shirai), yuzu (C. junos Tanaka), mochiyu (C. inflata Hort. ex Tanaka), and ponkan (C. reticulata Blanco cv. F-2426) inhibited the formation of NDMA much more effectively than other citrus oils. The inhibitory proportions of components of citrus essential oils such as myrcene, alpha-terpinene, and terpinolene were as high as 80%.

Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase.

Two novel highly water-soluble tetrazolium salts, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) were applied to the assay of superoxide dismutase (SOD). The superoxide anion generated by xanthine/xanthine oxidase (XO) reduced WST-1 and WST-8 to water-soluble formazans which exhibited absorbance maxima at 438 and 460 nm, respectively. The rates of reduction were linearly related to the XO activity, and reduction was inhibited by SOD. Complete inhibition by SOD of the reduction of both WST-1 and WST-8 was achieved, suggesting that these WSTs were not reduced with XO. WST-1 was found more useful than WST-8 because it had shown higher sensitivity which was apparently not dependent on the assay pH value in the range pH 8.0-10.2. These properties of WST-1 are ideal for the spectrophotometric assay of SOD in an aqueous system.

Glycolaldehyde Production from Ethylene Glycol with Immobilized Alcohol Oxidase and Catalase.

An enzymatic method for glycolaldehyde production from ethylene glycol was investigated using immobilized alcohol oxidase and catalase. Those enzymes were immobilized onto Chitopearl BCW 3501. When only alcohol oxidase was immobilized onto it, the apparent activity was 190 units/g in wet gel using methanol as the substrate. Tris-HCl buffer (1.5 M; pH 9.0) was selected based on a high stability of glycolaldehyde and a low production of glyoxal as a by-product. Under the optimum conditions, 0.97 M glycolaldehyde was formed from 1.0 M ethylene glycol and the ratio of glyoxal to glycolaldehyde was less than 1%.

Spectrophotometric assay for superoxide dismutase based on tetrazolium salt 3'--1--(phenylamino)-carbonyl--3, 4-tetrazolium]-bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate reduction by xanthine-xanthine oxidase.

XTT (3'-{1-[(phenylamino)-carbonyl]-3, 4-tetrazolium}bis(4-methoxy-6-nitro)benzenesulfonic acid hydrate) was reduced to a water-soluble product with an absorbance maximum at about 470 nm by superoxide anion generated by xanthine-xanthine oxidase (XO). The rate of XTT reduction was linearly related to XO activity and the reduction was inhibited by superoxide dismutase (SOD). A perfect inhibition of the reduction of XTT by SOD was achieved, suggesting that XTT does not interact with XO. The present XTT-based assay had a higher sensitivity than a conventional nitroblue tetrazolium-based assay by a factor of 2.5 at pH 10.2. This method was applicable to the SOD assay in the pH range 8.0-10.2.

Immunochemical approach to characterize post-translational modification of serum albumin using anti-glutaraldehyde-treated serum albumin antibodies.

Polyclonal antibodies against glutaraldehyde-treated rabbit serum albumin (pRSA) and human serum albumin (pHSA) were prepared from rabbit and mouse, respectively. Anti-pRSA antibody had the structural determinant depending on the polymerization process of RSA and showed only a weak cross-reactivity with the other glutaraldehyde-treated albumins. Anti-pHSA antibody (after adsorption of anti-HSA antibody) recognized only pHSA, but not HSA and the other treated albumins. The cross-reactivity of those antibodies was examined with albumins treated by other methods such as modification of glucose and fructose, carbodiimide, and transglutaminase. Among the, RSA and HSA modified with glucose and fructose had an affinity for each antibody and the reactivity depended on the extent of formation of the polymerized albumin. The results suggests that functional groups involved in cross-linking of albumin are important for formation of the cross-reactivity with the antibody and that a definite structure immunochemically similar to glutaraldehyde-treated albumin could be formed by the Maillard reaction.

Inactivation of Cu,Zn-superoxide dismutase by intermediates of Maillard reaction and glycolytic pathway and some sugars.

Human Cu,Zn-superoxide dismutase (SOD) was incubated with various intermediates of the Maillard reaction and glycolytic pathway (arabinose, glyoxal, glycolaldehyde, glyceraldehyde, glyceraldehyde 3-phosphate, and dihydroxyacetone) and some reducing sugars (sorbose, xylose, and ribose). The change of the activity and the molecular weight were measured and compared with that of SOD incubated with glucose or fructose. Sorbose, xylose, and ribose decreased the activity with a rate comparable to fructose. Site-specific and random fragmentation were observed upon the incubation with them. Arabinose showed a similar inactivation rate as glucose. The intermediates other than arabinose had a high inactivation rate. Especially, glyceraldehyde, glycolaldehyde, and glyoxal most strongly lowered the activity in a concentration-dependent manner and a significant inactivation was recognized even at 1 mM level. SDS-PAGE band patterns indicated that the inactivation by those carbonyl compounds occurred by both crosslinking and site-specific fragmentation of SOD.

Cluster Analysis by Measurement of Peroxidase and Esterase from Citrus Flavedo.

The zymograms of peroxidase (POD) and esterase (EST) from seventy-two kinds of Citrus fruits and the fruit of both Poncirus and Fortunella were obtained by polyacrylamide gel electrophoresis. It was found that there was little variance in the band pattern of POD and EST of the fruits harvested after September. There were at least three bands of POD and approximately ten of EST in each cultivar. The density of each band for EST was measured with a densitometer. There was a characteristic band for POD identifying pummelos. The band patterns of EST were also characteristic among each species or cultivar. Two kinds of ootachibana (Citrus otachibana Hort. ex Tanaka) were shown to be different cultivars from each other according to their EST zymograms and peel oil analysis. Cluster analysis based on EST was done and discussed together with POD.

Application of a microbial sensor for determination of short-chain fatty acids in raw milk samples.

A microbial sensor system, based on the use of immobilized Arthrobacter nicotiana and an oxygen electrode, was applied to determine free short-chain fatty acids in raw milk samples and the result was compared with gas chromatography (GC) and a titrimetric method. The sensor response was linearly related to the concentration of short-chain fatty acids obtained by GC (n = 10, r = 0.92) and to the total concentration of free fatty acids obtained by titrimetric measurement (n = 10, r = 0.78). This result suggests that the present microbial sensor can selectively determine free short-chain fatty acids in raw milk samples and may be useful as a very fast detection method of rancidity in milk.

Amperometric determination of amino group on a solid support using glutaraldehyde.

A simple amperometric method has been developed for the determination of amino groups on a solid support. The method is based on oxygen consumption during the reaction between the bifunctional reagent gluaraldehyde and the amino groups on the solid support. Oxygen consumption was monitored with a Clark oxygen electrode. AHSepharose 4B, Affi-Gel 102, and Amino-Celluofine were used as amino-terminal solid supports. The concentration of amino groups on each support was calculated from a calibration curve of the standard compound. When hexamethylenediamine was used as the standard, the concentration of amino groups on AH-Sepharose 4B and Affi-Gel 102 by the present method showed good agreement with that by the conventional method. In the case of Amino-Cellulofine, ethanolamine had to be selected as the standard. The relative standard deviation for six successive determinations of AH-Sepharose 4B was 3.2%; the polymerization of glutaraldehyde showed no effect on the result. The spectral property of AH-Sepharose 4B treated with glutataraldehyde showed no effect on the result. The spectral property of AH-Sepharose 4B treated with lutaraldehyde suggested that the reaction mechanism of the present method was based on the oxygen consumption during the formation of the pyridinium salt on the support. A comparative study of the present method with a colorimetric method with Traut's regent is also included.

Simultaneous determination of glucose, ethanol and lactate in alcoholic beverages and serum by amperometric flow injection analysis with immobilized enzyme reactors.

Glucose, ethanol and lactate were determined simultaneously in a flow injection system by using a parallel configuration of immobilized enzyme reactors. Hydrogen peroxide produced was monitored amperometrically at the potential of +0.65 V vs. Ag/AgCl. Linear relations between sensor responses and each species were observed in the ranges of 0.02-10 mM (glucose), 5 x 10(-4)-0.1% (v/v) (ethanol) and 0.005-1 mM (lactate) with correlation coefficients larger than 0.999 for each species. The relative standard deviations for 10 successive injections were 1.4, 0.5 and 1.1% for glucose (1 mM), ethanol (5 x 10(-3)% (v/v] and lactate (0.05 mM), respectively. Analysis of serum samples was performed with urate-eliminating reactors which were set just before each immobilized enzyme reactor. Interference of ascorbate in a serum sample was completely eliminated by using an ascorbate-eliminating reactor which was set before the sample injection valve. Application of the system to alcoholic beverages and control serum was described and the results were compared with those of free enzymatic, spectrophotometric analysis (F-kit or C-test method).