Masami Makita

Inihibitory effect of the ether extract of human feces on activities of mutagens: Inhibition of oleic and linoleic acids

An ether extract of normal human feces showed inhibitory effects on the activities of several mutagens in the Ames tests. By addition of the ether extract at an amount equivalent to 0.5 g of a sample of feces, the mutagenicity of 1.5 nmole of 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) on Salmonella typhimurium TA98 was completely inhibited. No killing of the bacteria was detected during this treatment. Other mutagens also subject to the inhibition were 2-amino-6-methyl-dipyrido[1,2-a:3,2-d]imidazole (Glu-P-1), 2-amino-9H-pyrido[2,3-b]indole (Glob-P-2), 2-amino-3-methylimidazo[4,5-d]quinoline (IQ), benzo[a]pyrene and aflatoxin B1. Apart from these mutagens, which require S9 for their activation, the direct mutagen prepared from Trp-P-1 by treatment with S9 was also inhibited by the fecal extract. The inhibitory principles in the fecal extract were fractionated by thin-layer chromatography on silica gel and were identified as oleic and linoleic acids. Whereas these unsaturated fatty acids showed strong inhibitory activities, saturated fatty acids, i.e, stearic and palmitic acids, did not exhibit any inhibition. Although the physiological significance of these effects of oleate and linoleate is yet to be elucidated, this finding has indicated that care must be taken in screening mutagens by the Ames tests to avoid false negatives resulting from the presence of unsaturated fatty acids in the system.

Simple and rapid determination of the herbicides glyphosate and glufosinate in river water, soil and carrot samples by gas chromatography with flame photometric detection

Abstract A rapid, selective and sensitive gas Chromatographic (GC) method for the simultaneous determination of the phosphorus-containing amino acid-type herbicides glyphosate (GLYP), its metabolite aminomethylphosphonic acid (AMPA) and glufosinate (GLUF) in environmental and food samples was developed. After extraction of the sample with water or sodium hydroxide solution, these compounds were converted into their N-isopropoxycarbonyl methyl ester derivatives and then measured by GC using a DB-1701 capillary column with flame photometric detection (FPD). The derivative preparation and GC analysis were accomplished within 20 min. The derivatives were sufficiently volatile and stable, and the FPD response was excellent. The detection limits of AMPA, GLYP and GLUF, at a signal-to-noise ratio of 3, were ca. 8, 12 and 20 pg injected, respectively. The calibration graphs for these compounds in the range 5–200 ng were linear and sufficiently reproducible for quantitative determination. This method could be successfully applied to river water, soil and carrot samples without a preliminary clean-up procedure, and AMPA, GLYP and GLUF could be measured without any interference from co-existing substances. The recoveries of these compounds in these samples were 91–106%.

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.

Analysis of lipoic acid in biological samples by gas chromatography with flame photometric detection

A selective and sensitive gas chromatographic method for the analysis of lipoic acid in biological samples has been developed. After base hydrolysis of the sample, the liberated lipoic acid was converted into its S,S-diethoxycarbonyl methyl ester derivative and measured by gas chromatography using a DB-210 capillary column and a flame photometric detector. The calibration curve was linear in the range 20-500 ng, and the detection limit was ca. 50 pg injected. The best hydrolysis conditions for the biological samples were obtained by using 2 M potassium hydroxide containing 4% bovine serum albumin at 110 degrees C for 3 h. Using this method, lipoic acid in the hydrolysate could be selectively determined without any interference from matrix substances. Analytical results for the determination of lipoic acid in the mouse tissue and bacterial cell samples are presented.

Determination of total plasma homocysteine and related aminothiols by gas chromatography with flame photometric detection

A selective and sensitive method for the determination of total homocysteine (Hcy) and related aminothiols, such as cysteine (Cys) and cysteinylglycine (CysGly), in plasma samples by gas chromatography (GC) has been developed. After reduction of the sample with sodium borohydride, the liberated Hcy and other aminothiols were converted to their N,S-diisopropoxycarbonyl methyl ester derivatives and measured by GC with flame photometric detection using a DB-17 capillary column. The calibration curves were linear over the range 0.5-10 nmol for Hcy and CysGly, and over the range 5-100 nmol for Cys, and the correlation coefficients were above 0.996. Using this method, total plasma Hcy, Cys and CysGly could be directly analysed without prior clean-up of the sample and without any interference from coexisting substances. Overall recoveries of Hcy and other aminothiols added to plasma samples were 95-106%. Analytical results for the determination of total plasma Hcy, Cys and CysGly from normal subjects are presented.

Inhibition of the mutagenicity of cooked-beef basic fraction by its acidic fraction

By using the Salmonella/microsome system, it was found that the activity of mutagens present in the basic fraction of cooked-ground-beef was completely suppressed by addition of the acidic fraction obtained from the cooked-beef. The suppression was ascribable to the presence of oleic acid in the acidic fraction. This finding indicates that no, or diminished, mutagenicity would be found in materials containing fat.

Determination of selenocyst(e)amine, selenocyst(e)ine and selenomethionine by gas chromatography with flame photometric detection

Abstract A selective and sensitive method for the determination of selenocyst(e)amine, selenocyst(e)ine and selenomethionine (Se-Met) by gas chromatography (GC) was developed. Selenocystamine and selenocystine were reduced to selenocysteamine (Se-CYE) and selenocysteine (Se-Cys), respectively, by adding sodium tetrahydroborate prior to derivatization. Se-CYE, Se-Cys and Se-Met were converted into N,Se-isopropoxycarbonyl (isoPOC), N,Se-isoPOC methyl ester and N-isoPOC methyl ester derivatives, respectively, and determined by GC with flame photometric detection (FPD) using a DB-17 capillary column. These derivatives were sufficiently volatile and stable, giving single and symmetrical peaks, and provided an excellent FPD response. The detection limits of Se-CYE, Se-Cys and Se-Met were ca. 1.4, 1.0 and 1.4 pmol injected, respectively. The calibration graphs for these selenium compounds were linear in the range 1-20 nmol and sufficiently reproducible for quantification.

Determination of aromatic amines as their N-dimethylthiophosphoryl derivatives by gas chromatography with flame photometric detection

Abstract A selective and sensitive method was developed for the determination of aromatic amines by gas chromatography (GC). Aromatic amines were converted into their N-dimethylthiophosphoryl derivatives and measured by GC with flame photometric detection using two-connected fused-silica capillary column containing DB-1 and DB-17, respectively. The derivatives were sufficiently volatile and stable to give single symmetrical peaks. The calibration graphs for aromatic amines in the range 25–2000 ng were linear and the detection limits at a signal-to-noise ratio of 3 were ca. 30–100 pg injected. This method was successfully applied to smoke samples without prior clean-up. Overall recoveries of aromatic amines added to cigarette smoke samples were 90–108%. Analytical results for the contents of aromatic amines in combustion smokes of various samples are presented.

Selective determination of volatile N-nitrosamines by derivatization with diethyl chlorothiophosphate and gas chromatography with flame photometric detection

A selective and sensitive method was developed for the determination of volatile N-nitrosamines by gas chromatography (GC). After denitrosation of N-nitrosamines with hydrobromic acid, the resulting secondary amines were converted into their N-diethylthiophosphoryl derivatives and then measured by GC using a DB-1701 capillary column with flame photometric detection. The calibration graphs for N-nitrosamines in the range 0.05-1 nmol were linear and sufficiently reproducible for quantitative determination. This method was successfully applied to cigarette smoke samples without prior clean-up. N-Nitrosamines and secondary amines were completely separated by extraction with diethyl ether containing 25% 2-propanol. Overall recoveries of N-nitrosamines added to cigarette smoke samples were 83-110%. By using this method, N-nitrosamines in these samples could be determined without any interference from coexisting substances. Analytical results for the contents of N-nitrosamines and secondary amines in mainstream and sidestream smokes of several cigarettes are presented.

Determination of amino acids in human serum by capillary gas chromatography

A simple and rapid method for the determination of serum amino acids by gas chromatography (GC) has been developed. Following deproteinization of serum with perchloric acid, free amino acids in the supernatant were converted into their N(O,S)-isobutoxycarbonyl methyl ester derivatives and measured by GC with flame ionization detection using a DB-17 capillary column. All the derivatives of the 22 protein amino acids were completely resolved as single peaks within 9 min by GC. The calibration curves were linear in the range 0.2-50 micrograms of each amino acid, and the correlation coefficients were above 0.998. By using this method, serum amino acids could be directly analysed without prior clean-up procedure such as ion-exchange column chromatography except for deproteinization of the samples, and without any interference from coexisting substances. Overall recoveries of amino acids added to serum samples were 88-108%. Analytical results for serum amino acids from normal subjects are presented.