A simple method for determination of vitamin A and β-carotene by highspeed liquid chromatography was established. In this analytical method, the sample to be tested, added with pyrene as an internal standard, was saponified and unsaponifiable matter was extracted once with 80ml of ether. The residue obtained by evaporation of ether was dissolved in a solvent of the same composition as that of the mobile phase for the chromatography and this solution was injected. A wavelength variable detector was used to detect vitamin A at 318nm, and then the wavelength was changed to 453 nm to detect β-carotene. From the chromatogram obtained, quantities of vitamin A and β-carotene were calculated from the ratio of peak heights, using pyrene as the internal standard. In crude oil, the values showed a slight positive error due to interference of substances originating in the unsaponifiable matter of oil. In the recovery test with purified oil, the rate was 96.5-103.4% for vitamin A and 93.5-95.5% for β-carotene. There was no significant difference between individuals carrying out the analysis. This method was applied for the analysis of commercial margarine and butter, and comparison of the values obtained with the known analytical values showed that the values for vitamin A and β-carotene by this method were satisfactory, indicating usefulness of this method of analysis.
Inactivation of chymotrypsinogen in the presence of xanthene dyes such as rose bengale, phloxine, erythrosine and eosine was observed through the irradiation of light, but not in the dark. The extent of inhibition was in the following order; rose bengale>phloxine>erythrosine>eosine. Interaction of xanthene dyes with chymotrypsinogen were also examined by the spectrophotometric methods. In the presence of chymotrypsinogen, the absorption maxima of the dyes did not shift at pH 6.97, 7.99 and 10.0, but shifted to longer wavelength at pH 4.73 and 5.78. The binding numbers and the binding free energies were calculated from the difference spectra for the mixtures of the dyes and chymotrypsinogen which were incubated at 37°C for 15min. In comparison the both indexes of rose bengale to chymotrypsinogen in the dark with those in the light, they were not so increased in proportion to the inactivation of chymotrypsinogen. The order of binding capacities and affinities was rose bengale>phloxine>erythrosine>eosine at pH 4.77, regardless of the conditions with and without the irradiation. It was assumed that chymotrypsinogen would be inactivated in the presence of xanthene dyes by the photo-effect, not by the binding capacities and/or the affinities.
Residual vinyl chloride monomer (VCM) in poly-vinyl chloride (PVC) products was measured by mass-fragmentgraphy. The sensitivity of this method was more than twenty times the GC method, and VCM could be analyzed without any prevention of or misidentification with other compounds having near retention times. Fifteen samples of commercial PVC products such as bottle for food were dissolved in tetrahydrofuran and VCM was analyzed by this method. The concentration of VCM in one sample of a bottle which prepared for edible oil in 1974 was 20.4ppm, and the values for the others were less than 1ppm; 1.0-0.05ppm for 6 samples and non-detected (<0.05ppm) for 8 samples.
It was found that the release of VCM in PVC products was accelerated by heating of the sample with solvent in a sealed container. Twenty to five hundred mg of cut plate sample of PVC was set in a test tube of about 10ml volume and stoppered tightly by a silicone rubber stopper, after addition of 0.1ml of solvent such as chloroform, and heated in an oven at 100°C for 30 minutes. The released VCM in the tube was immediately injected into GC or GC-MS apparatus by a pressure-lock gas sylinge and the VCM was analyzed. The result was equal to that of the tetrahydrofuran solution method. By this method, VCM at a level of 0.01ppm could be detected even with gas chromatography and shorten the operation time.
The effect of various organic acids, such as citric acid, tartaric acid, malic acid, tricarballylic acid and so forth, on the rates of nitrosation of hydantoic acid (HA) and methylurea (MU), which were ureido compounds, was studied. These organic acids accelerated the rate of nitrosation of the ureido compounds. Extents of accelerating effect of the organic acids were greater in the compounds which have more carboxyl groups or hydroxy groups. The rate of nitrososarcosine formation from creative, one of guanido compounds, and nitrite was accelerated by citric acid, tartaric acid and sodium thiocyanate. The reaction of HA and sodium nitrite in presence of citric acid was first order with respect to each substance, and the following equation was set up: rate=k1[HA][nitrite]+k2[citrate][HA][nitrite]. The rate of carboxymethyl nitrosourea (CMNU) formation from HA and nitrite was increased with lowering pH, and this pH dependence was not remarkably affected in the presence of citric acid.
Distribution of cadmium (Cd) in the mother-bodies, its transference to the fetus, and its subcellular distribution in the placentae were investigated in pregnant rats of gestational day 18 following a single or repeated subcutaneous injection of CdCl2 at various periods of gestation. 1) Most of Cd injected at any period of gestation was present in the liver and kidney, in which Cd contents were 36.5-39.9% and 1.5-3.3% of the administered dose, respectively. In the mother-bodies, higher concentration of Cd was found in the spleen, heart and uterus than in the blood. 2) Transference of Cd to the fetus after parenteral injection of CdCl2 was not so remarkable, but a small amount of Cd was found in the amniotic fluid. It is suggested that Cd may be transferred in part to the fetus through the placentae. 3) Concentration of Cd in the placenta after a single injection of CdCl2 on gestational day 15 was extremely high, as compared with that after the injection on gestational day 0. The repeated injection of CdCl2 caused higher concentration of Cd in the placenta than the single injection, when the total amounts of both doses were the same. 4) In the placenta, Cd was mostly located in the nuclear and supernatant fractions. In the supernatant fraction obtained from the placenta after injection of CdCl2 on gestational day 15, Cd was predominantly associated with the protein fraction with molecular weight of 20, 000-30, 000 on gel-filtration chromatography.
The gastro-intestinal absorption of N-nitrosodimethylamine (NDM) and its precursors in guinea-pigs were studied. NDM, dimethylamine (DMA) and nitrate were quite stable in the contents of stomach and small intestine when they were incubated in vitro at 37°C for 60min. Nitrite was stable in the contents of small intestine, but not in those of stomach. DMA and nitrate disappeared from the ligated small intestine, but not from the ligated stomach. Nitrite rapidly disappeared from the ligated stomach, and the rate of disappearance was more rapid than that of decomposition. The disappearance curves of NDM from the ligated stomach and small intestine were monoexponential. NDM rapidly disappeared from the small intestine, and slowly from the stomach.
As a part of the study on safety evaluation of detergents, the hemolytic activity and the effect on glycolysis in human erythrocytes were investigated. Fifty per cent hemolysis by various detergents occurred in the range from 7 to 73ppm in low blood concentration, but the hemolytic activity in high blood concentration (50v/v%) was decreased to about one-hundredth. The order of hemolytic activity was almost parallel to the lowering activity of surface-tension. In general, the hemolysis by anionic detergents was more potent than that of nonionic ones. The glycolysis in erythrocytes was not influenced by treatment with various detergents at the concentration of one-fourth of 50% hemolytic value. However, the glycolysis in burst erythrocytes was inhibited from 50 to 70% by the anionic and stimulated from 30 to 100% by the nonionic, at 10-3M concentration of detergents. From the studies with some glycolytic enzymes, it was suggested that the inhibition of glycolysis by anionic detergents was due to the formation of the enzyme–detergent complex, and the stimulation by nonionic detergents was attributed to the stimulation of aldolase activity.
Experiments were designed to establish the method for viable cell counts of bifidobacteria in the fermented milk. Results obtained are as follows: 1) The solution containing 0.1% of yeast extract or corn steap liquor was superior for the viable cell counts of bifidobacteria as the diluents of samples for the serial dilution to the physiological saline usually used in the enumeration of lactic acid bacteria. 2) Modified Rogosa's and BL agar were demonstrated to be the best media for the viable cell counts of both bifidobacteria and lactic acid bacteria in the fermented milk, but B. C. P. plate count agar which were generally used for the enumeration of the latter, was not suitable for the determination of bifidobacteria. 3) Anaerobic cultivations are essential for the measurement of viable cells of bifidobacteria and either of anaerobic jar method, Gas Pak anaerobic jar or Pouch method is adapted for this purpose. However, there was little difference in superiority among them. 4) Bifidobacterium formed colonies 0.5 to 3.0mm in diameter after the cultivations at 37°C for 48 to 72hr under the conditions described above. 5) Differential calculation of viable cells of bifidobacteria and lactic acid bacteria was completely established from the mixed cultures of the two or three species using modified Rogosa's selective agar together with B. C. P. plate counts agar. Because Bifidobacterium could grow on the Rogosa's selective agar, but the growth of lactic acid bacteria was inhibited. 6) The detection of bifidobacterial colony decreased remarkably, when both of the Gas Pak anaerobic jar and Pouch method generally used as the simple techniques for anaerobic cultivations, were applied to their viable cell counts with the modified Rogosa's selective agar.
Skipjack (Katsuwonus pelamis) and seerfish (Scomberomorus niphonius), caught in Kishu, 57 and 70cm fork length, respectively, were determined selenium and total mercury contents in different tissues. Selenium contents in the ordinary muscle, dark muscle, liver, spleen and kidney of skipjack were 0.40, 3.2, 9.2, 2.4 and 2.4μg/g, respectively, and in the same tissues of seerfish were 0.33, 0.78, 2.7, 3.8 and 3.7μg/g, respectively. Ventral portion contained more selenium than dorsal portion in the ordinary muscle of the two fishes. Total mercury contents in the ordinary muscle of skipjack and seerfish were 0.32 and 0.06μg/g, which uniformly distributed in each muscle. In skipjack the ordinary muscle showed the highest total mercury contents among the all tissues, but did not in seerfish. Total mercury contents in the ordinary muscle of the two fishes, increased with fork length, but selenium contents either remained constant or decreased. The similar tendency was observed in yellowfin tuna (Neothunnus albacora). Contents of the both elements were compared amongg the three fishes, mentioned above, mackerel (Pneumatophorus japonicus) and red sea bream (Pagrus major) of nearly same age. Total mercury contents in the ordinary muscle of these five species were significantly different, while selenium contents were not. However, contents of the both elements in the liver were remarkably different.
A sensitive method for the determination of alkyl mercury in distillate gained by steam distillation was described. The wet samples (5-50g) were put into a 500ml distilling flasks and steam distilled after adding 40ml of 6N hydrochloric acid and 40g of sodium chloride. The distillate was put into 10ml of 3N hydrochloric acid, being about 180ml of final volume. The distillate was transferred into a 200ml volumetric flask and dilute with distilled water. A 50ml portion of distillate, 20ml of distillated water, 2ml of 0.4% cupric sulfate solution and 20ml of 40% sodium hydroxide solution were transferred into a reaction vessel and mixed togather by slightly shaking, then mercury vapor generated by addition of 10ml 5-% stannous chloride solution determined by flameless atomic absorption spectrophotometer equipped with integration apparatus. By this procedure herein described, alkyl mercury in a wide variety of fish and shellfish could be sensitively determined. The data have shown quantitative recovery of alkyl mercury added to a variety of samples. The coefficient of variation for repeated determinations on a black bream meat sample was 1.9%. If the flameless atomic absorption spectrophotometer was not equipped with integration apparatus, alkyl mercury in distillate could be determined from peak height (conc. method) as sensitive as the integration method mentioned above, by raising reaction temperature of reduction to about 40°C.