Previously, we proposed a standard approach for quantification of the intensities of mouse anaphylactic responses using the fall in blood pressure (mouse anaphylactic hypotension test; MAHT) after treatment with food allergens. In the present study, we examined the effects of several adjuvants in an attempt to shorten the period required for the test. We compared Freund's incomplete adjuvant (FIA) and aluminum hydroxide gel (Alum) as adjuvants. Ovalbumin was easily adsorbed on Alum, but hen egg-white lysozyme was poorly adsorbed. The adsorption of proteins on Alum was dependent on molecular weight and isoelectric point. The protein's molecular form and surface electric charge might also influence adsorption on Alum. Accordingly, Alum appeared to be unsuitable as an adjuvant for this method. Therefore we mixed 20mg/mL Alum and FIA (1:1) (Alum+FIA), and found that allergic reaction intensities with Alum+FIA were stronger than those with either Alum or FIA alone. Moreover, Alum could also be utilized for evaluating allergenicity of egg-white, and allergic reaction intensity with Alum+FIA was stronger than that with FIA alone. The allergenicity of boiled egg-white was weaker than that of raw egg-white. Boiling may convert water-soluble into water-insoluble substances in egg-white, or antigenic determinants may be partly denatured.
A simple and rapid method has been developed for the determination of taurine in foods by HPLC with an on-column fluorescence derivatization technique. Taurine was extracted from foods with water, and the crude extract was deproteinized with Carrez solution, followed by purification on a Bond Elut SCX cartridge column. Taurine was chromatographed on a polymer column with a mobile phase of acetonitrile and alkaline borate buffer solution containing o-phthaladehyde and 2-mercaptoethanol as derivatizing reagents. The average recoveries were greater than 90% for taurine added to various kinds of foods, such as milk products, shellfish, chicken egg and honey. Application of the method to the foods resulted in the detection of taurine in egg (1.26±0.15mg/100g) and honey (0.34±0.08mg/100g), in which taurine had not previously been found.
A rapid analytical method for the determination of ochratoxin A (OCTA), without using chloroform or an immunoaffinity column, in coffee beans and cereals such as rice, wheat, barley and corn was developed. OCTA was extracted from spiked food samples with acetonitrile-1% H3PO4 (99:1) and cleaned up using cartridge anion exchange column (Bond Elut DEA) chromatography. The test solution was chromatographed on a Capcell Pak C8 column with acetonitrile-water-acetic acid (40:58:2). OCTA was detected using a fluorescence detector (excitation: 335nm; emission: 465nm). Recoveries from samples spiked with 10ng/g OCTA and 50ng/g in coffee beans, unpolished rice, wheat, barley and corn averaged 93.9±3.9% and 96.1±3.5%, respectively. The detection limits corresponded to 0.05ng/g in green coffee beans, roasted coffee beans, unpolished rice, wheat, barley and corn.
Cardiac glycosides in “Moroheiya” (C. olitorius) and its products were analyzed as their aglycone, strophanthidin (SP), by reverse-phase HPLC with UV detection. Methanol extracts of “Moroheiya” samples were hydrolyzed with 1mol/L HCl and cleaned up with a Bond Elut Silica cartridge. Well-matured seed of C. olitorius contained large amounts of cardiac glycosides in the range of 4.1-6.6mg/g (average 5.43mg/g) as SP. The pod also contained a small amount of cardiac glycosides (0.24μg/g as SP). However, no cardiac glycosides were detected in the fresh “Moroheiya” (including green leaf, green stem, shoot and pod), which has been used in Japan as a vegetable, in dried health tea products and in health food products. Spectroscopic data suggested that there were at least two cardiac glycosides in C. olitorius seed.
Three thousand seven hundred and eighty samples were taken from nostril anteriors, fingers, hands, face, hair, pierced ear lobe, etc. of 739 trade school students by the subjects themselves between April 1996 and March 1997 and tested for the incidence of Staphylococcus aureus (S. aureus). The incidence of S. aureus from nostril anteriors was 12.6% in a total of 1, 130 strains examined. The predominant types of S. aureus in nostril anteriors tested were coagulase type VII (45.2%), including enterotoxin type B (40.3%), followed by enterotoxin type A (25.4%). Methicillin-resistant S. aureus (MRSA) was not detected. In 3 tests within 1 year on S. aureus-carrier students, 76.7% of the students retained S. aureus and almost all of such students showed the same coagulase and enterotoxin types. The coincidence between the S. aureus isolated from the carrier students and that from their families was 57.9% for the coagulase type and 100% for the enterotoxin type.
The tissues levels of oxytetracycline were determined after oral administration (50mg/kg) in cultured eel (Anguilla japonica) and ayu (Plecoglossus altivelis). The water temperature was adjusted to 28°C for eel and 18°C for ayu. Oxytetracycline levels were determined by HPLC. Concentrations of oxytetracycline were in the order of bone>liver>skin>muscle>serum for eel and liver>bone>skin>muscle>serum for ayu. The elimination half-lives of serum, muscle and liver were 4, 6 and 11 days for eel, and 5, 7 and 6 days for ayu, respectively. The elimination times of serum, muscle and liver were calculated to be 4, 5 and 25 days for eel, and 10, 14 and 24 days for ayu, respectively. However, the elimination half-lives and elimination time of skin and bone could not be calculated because oxytetracycline residues persisted in skin and bone in both fish species, and no elimination phase could be recognized up to 30 days. Residual oxytetracycline was reduced by 70-80% in muscle and liver by the usual methods of cooking (boiling, baking and frying), whereas the reduction in bone was only 30-50%.
Thirty species of shellfish were collected at Fukue Island, Nagasaki Prefecture in July 1995 through October 1996, and screened for paralytic shellfish poison (PSP). Six species were found to be toxic: Pecten albicans (scallop), Chlamys farreri (scallop), Septifer virgatus (mussel), Pinna bicolor, Arca boucardi and Pseudochama retroversa. In both scallops, the digestive gland was most toxic, with the highest score of 133.8 MU/g in P. albicans. The toxin profile of P. albicans featured the dominant presence of gonyautoxins (GTXs) in 1995. In 1996, however, low-toxicity components such as the C (PX) group were major, as was the case in the profile of C. farreri. In this connection, the mossworm adherent to shells of C. farreri contained a low level of PSP, whose major components were decarbamoyl GTX 2 (dcGTX2) and GTX2, along with dcGTX3 and GTX3, differing clearly from the components of C. farreri.
This study was carried out to determine the in vitro binding of heterocyclic aromatic amines (HAAs), IQ, MeIQ, Trp-P-1, and Trp-P-2, to low molecular weight cellulose using HPLC assay and bacterial mutagenicity assay. Eight types of low molecular weight cellulose (degree of polymerization (DP): 125-180, particle size: 8-80μm, settling volume in water (SV): 3.2-6.6mL/g) were used in this study. All types of DP 180 cellulose showed higher adsorptive capacity for IQ, MeIQ, Trp-P-1 and Trp-P-2 than the DP 125 and DP 130. The 4 HAAs showed no significant difference in adsorption by cellulose from 10 to 80μm in particle size. In the case of 2 types of cellulose with the same DP (180) and particle size (40μm) but different SV (5.4mL/g, 4.0mL/g), Trp-P-1, Trp-P-2 and MeIQ showed no difference in adsorption but IQ was adsorbed at a higher rate (1.3 times) by the cellulose of larger SV. Highly hydrophobic compounds among the 4 HAAs (Trp-P-1 and Trp-P-2) were adsorbed by cellulose in large quantities, strongly and quickly. The results of the two assays seemed to show similar patterns in the adsorption of the 4 HAAs by the 8 types of cellulose. Based on the HPLC assay, Trp-P-1 and Trp-P-2 were adsorbed by the 8 types of cellulose at rates of 81-95% and 71-95%. Similarly, the numbers of revertants per plate induced by Trp-P-1 and Trp-P-2 treated with these celluloses were 0.7-2.1% and 0.4-5.0% of the control, respectively. From these results, it has been clarified that low molecular weight cellulose with a DP about 24 to 80 times smaller than the native value can adsorb HAAs.
The effect of hexametaphosphate (HP), a long-chain polyphosphate known to have an antibacterial (bacteriostatic) action on Staphylococcus aureus through its action on cytoplasmic membranes, was studied. Using spheroplasts prepared from this bacterium, HP was observed to stimulate the leakage of nucleic acid-related substances, nucleic acids and proteins from the spherotoplasts in 1.2mol/L sucrose solution. In addition, using cytoplasmic membranes isolated from the cells, it was found that HP slowly released proteins in small amounts and rapidly released magnesium in large amounts from the membranes. The results indicated that HP acts on the cytoplasmic membranes. These findings suggest that the influence of HP on the cytoplasmic membranes of S. aureus plays an important role in its action on this bacterium.
This study showed that chemiluminescent (CL) assay is superior to Giemsa staining assay or MTT reduction assay in sensitivity and rapidity for detection of the cytotoxic effects of four compounds (phenobarbital sodium, theophyllin, propranolol and thioridazine). Furthermore, HepG2 cells were found to be more suitable for CL assay than intestine 407 cells, being more susceptible to the toxicity of the four compounds.
A simple GC method with a capillary column was developed for simultaneous determina tion of 6 carrier solvents (ethanol, propylene glycol, glycerin, triacetin, diethylene glycol monoethyl ether and 1, 3-butylene glycol) in flavor preparations. Samples were diluted with n-propanol, and 1μL aliquots were injected into a GC apparatus. No further treatments were carried out to remove interfering substances. Out of 96 flavor preparations, ethanol was found in 68, propylene glycol in 31, glycerin in 18, and triacetin in 12.
Puffer fishes are commonly found in Hong Kong waters. For this study, we collected and identified six species of local puffers, Takifugu niphobles (Jordan and Snyder), Takifugu alboplumbeus (Richardson), Chelonodon patoca (Hamilton-Buchanan), Lagocephalus spadiceus (Richardson), Takifugu ocellatus (Osbeck) and Diodon holocanthus (Linnaeus). The first three species were confirmed to be toxic (6-120 mouse units (MU)/g) by mouse bioassay. To isolate tetrodotoxin (TTX) from a common local puffer, Takifugu niphobles, two hundred grams of viscera (ovary, intestine and liver) was minced and extracted with acidic methanol. The crude toxin obtained was further purified by Sephadex G-50-50 gel filtration and Whatman CM-52 cation exchange column chromatography. Purification afforded two unidentified toxins, unknown 1 and unknown 2, which gave single fluorescent spots under UV irradiation (366nm) with Rf values of 0.80 and 0.54, respectively, in thin layer chromatography (TLC). The electrospray ionization mass spectra (ESI/MS) of unknowns 1 and 2 had their molecular ion peaks (M+H)+ at m/z 320 and 255, respectively. Unknown 1 was concluded to be TTX, whereas unknown 2 was probably a derivative of TTX, whose exact identification would require further chemical analyses.