Citrinin is a toxic metabolite produced by several Penicillium spp. and a few Aspergillus spp. Citrinin is nephrotoxic and has been implicated in disease outbreaks associated with moldy rice. A reversed-phase ion-pair high-performance liquid chromatographic method was developed for determining citrinin. Citrinin was extracted from a grain sample, then the extract was cleaned up by partitioning with solvents, and an aliquot was injected into a stainless steel column containing Partisil-10 ODS-3. The mobile phase was 47% ethanol/PIC-A (tetrabutylammonium phosphate 0.005M) and 53% water/PIC-A. Detection was achieved by ultraviolet absorbance measurement at 254nm. Retention time and peak hight were highly reproducible. The relationship between peak height and quantity injected was linear over a range of 1-103ng. The detection limit of the method is 20ng/g. Recovery of citrinin from a grain sample spiked at the level of 0.25μg/g was 66%. The method was applied to the analysis of commercial rice and barley samples. Citrinin was not detected in 10 samples examined.
The mode of the combined effect of hexametaphosphate (HP) and glycerol monocaprate (MC10) was investigated by using E. coli JE 1011 (JE) and its NS mutants, which were found to have incomplete lipopolysaccharides. Though HP or MC10 alone had little antibacterial activity against JE, the combined use of the two substances showed much higher activity against the bacterium. In the case of combined use, polyphosphates having higher molecular weight or monoglycerides having longer carbon chain-length of fatty acid (within the limits of C6 to C12) showed stronger antibacterial activity than those having lower molecular weight or those having shorter chain fatty acid. The spheroplasts prepared from JE were lysed by monoglycerides. The cell lytic activity of monoglycerides was highest in the case of MC10, followed by MC8 and MC6 in order of decreasing activity. The activities of MC12 and MC14 were very low. We found that HP acted on the cell envelope of JE first and then MC10 acted on the altered envelope. It was confirmed by using JE and its NS mutants that the resistance of JE to HP and MC10 involved the outer membrane of the cell. The mode of the combined effect of HP and MC10 differed from that of the effect of HP and cholate.
The target sites of glycerol monocaprate (MC10) against Escherichia coli JE 1011 (E. coli JE) were elucidated by using succinate dehydrogenase (SDH), a membrane-bound enzyme of the cytoplasmic membrane. Succinate dehydrogenase of E. coli JE was activated in vivo by MC10 but that of the cell envelopes prepared from the bacterium was inhibited by it. In addition, we used the NS-mutants (NS-3, NS-4) which were found to have incomplete LPS and carried out similar experiments. In vivo, SDH of NS-3 was activated by MC10 but that of NS-4 was inhibited by it. However, SDH activities of the cell envelopes prepared from these NS-mutants were inhibited by MC10. From these results, we reduce that MC10 may act on the outer membrane of E. coli JE, stimulating the permeation of substrates and apparently activating the SDH of the cell in vivo. Thus, it is likely that the first target site of MC10 is on the outer membrane of E. coli JE.
Biochemical effects induced by intraperitoneal injection of mushroom extracts into mice were investigated in six species of mushroom which were suspected to cause cholera-like symptoms in man. They were Amanita verna, Amanita virosa, Amanita citrina, Amanita pseudoporphyria, Amanita abrupta and Galerina fasciculata. The extracts of all the species of mushroom examined decreased the contents of liver glycogen and increased the activities of serum glutamate pyruvic transaminase (GPT). The blood glucose level was decreased by the administration of extracts of five species (not Amanita pseudoporphyria). Among the poisonous mushrooms examined, Amanita abrupta exhibited the greatest effects in various other clinical chemical tests.
The water-soluble fraction of soybeans was extracted with 0.01M Tris-hydrochloric acid buffer (pH 7.4). The high molecular weight fraction (F-I, MW.>10, 000) was separated from the soluble fraction by gel filtration on Sephadex G-50. The chemical forms of copper (Cu) in F-I were investigated. F-I was separated into five components by chromatography on a Sephadex G-100 column (2.0×70cm). Most of the endogeneous copper in F-I was eluted in the highest molecular weight fraction (F-IA, MW.>100, 000), the major component in F-I. When copper was added exogeneously to F-I, further copper could bind with F-I and over half the bound copper was found in F-IA. Copper-binding affinities of F-I and F-IA were determined by equilibrium dialyses. The dissociation constants of F-I and F-IA for copper were calculated to be 2.3×10-5M and 2.0×10-5M, respectively, by fitting the Scatchard equation to the copper binding equilibrium curves. Maximal binding amounts of copper with F-I and F-IA were estimated to be 6, 000 and 5, 700ppm, respectively. Artificial [F-I]-Cu complex containing a high level copper was prepared by the addition of copper to F-I. The copper content of the artificial complex was almost constant (2, 700ppm) in several experiments. The stability of this complex were studied by equilibrium dialyses against EDTA-2Na, 2-mercaptoethanol (2-ME), organic acids (6 kinds), amino acids (15 kinds) or metal ions (10 kinds). The artificial complex was unstable to EDTA-2Na, histidine, Zn2+, Hg2+ or Cd2+. On the other hand, the complex was found to be stabilized by compounds having SH groups: 2-ME, cysteine and glutathione (reduced form).
Int he determination of microamounts of hydrogen peroxide in foods containing a large amount of lipid peroxide by the 4-aminoantipyrine (4-AA) colorimetry, the values determined tend to be excessive. To solve this problem, the removal of the interfering substances was studied by using linoleic acid, which readily forms lipid peroxide on UV irradiation, as a model compound. The interfering substances in linoleic acid irradiated with UV light were removed by a washing procedure with ethyl ether to improve the accuracy of 4-AA colorimetry; hydrogen peroxide was not decomposed. By gas chromatography with a flame ionization detector and by thin layer chromatography, two kinds of interfering substances were detected in the ethyl ether after the washing procedure. The present method was applied to foods which showed relatively large amounts of hydrogen peroxide in the 4-aminoantipyrine colorimetry, and the values determined by the present method were lower, being in good agreement with those found by the oxygen electrode method.
A high performance liquid chromatographic procedure (HPLC) has been developed which is fast, specific and reliable over a wide range of sugar concentrations in foods. The samples can be prepared for analysis within 1.5 hours and the following sugars can be separated in less than 20 minutes: fructose, glucose, sucrose, maltose and lactose. Sugars in food samples containing much starch and protein were extracted for 15 minutes with 80% ethanol and those in other samples were extracted with water. The sugars were separated on a column packed with Nucleosil-5NH2 (5μm) by using acetonitrile-water (70:30, v/v%) as a mobile phase. The coefficient of variation was less than 7.3% for food samples and the recovery was in the range of 93.1 to 103.0% for five sugars. The sugar determination by HPLC is closely related to that by gasliquid chromatography (GLC). Because sorbitol is not separated from glucose by HPLC, foods containing sorbitol must be analyzed by GLC.
Quantitation of histamine in fishes and fish products was studied by an HPLC-post-column derivatization method. Histamine extracted from samples was separated on a cation exchange column with 0.5M citric buffer (pH 6.4) as a mobile phase, and then derivatized with 0.1% o-phthalaldehyde solution (in borate-KOH buffer, pH 12). o-Phthalaldehyde-histamine complex was determined with a fluorescence detector (excitation at 360nm, emission at 455nm). Commercial fishes (8 samples), fish products (6 samples) and fresh fishes (4 samples) caught in Osaka Bay were analyzed by this method. Among commercial fishes, 5.5ppm of histamine was detected in a jack mackerel. In the case of fish products, high levels of histamine were detected from two dried Japanese pilchards (1050ppm, 382ppm). No trace of histamine was detected in fresh fishes. However, when a fresh mackerel was left to stand at room temperature for 48 hours, the level of histamine in the mackerel increased to 28.4ppm (24 hours) and 1540ppm (48 hours).
A high performance liquid chromatographic method for the simultaneous determination of oxolinic acid (OA), nalidixic acid (NA), and piromidic acid (PA) in fishes has been developed. The extraction and clean-up methods previously reported for analysis of NA could be applied to analysis of OA and PA. Except for the wavelength of UV detection, liquid chromatography was performed under the same conditions as used for analysis of NA. The UV detector was operated at 335nm in order to avoid interference in the chromatograms of fish extracts. Each compound was separated clearly in approximately 15min. The calibration curves were linear in the range from 1 to 50μg/ml. The recoveries of OA, NA, and PA added to fishes were 87.5-90.0, 85.0-86.3 and 74.5-77.2% and the coefficients of variation were 0.6-2.2, 0.8-2.0 and 0.5-2.1%, respectively. The detection limits of OA, NA, and PA were 0.03, 0.04 and 0.1μg/ml, respectively.