Norio Shibuya

Determination of Free Fatty Acids in Human Bile by High-Performance Liquid Chromatography

We developed a high-performance liquid chromatography (HPLC) method for free fatty acids (FFAs) analysis in bile. In this method, FFAs were extracted from bile in a single step using an Isolute™ ODS cartridge, derivatized with 9-anthryldiazomethane (ADAM). ADAM was chosen because of its high reactivity with carboxylic acid at room temperature. Then, HPLC was used for separating and quantifying FFAs. This method proved to be simple and time-saving. The mean recovery of FFA added to human gallbladder bile was 97.6%, and the detection limit was 100–250 pg. Using this method, we determined FFA concentrations in the gallbladder bile of 11 gallstone patients. The mean concentration of total FFA was 0.61 (SD = 0.41) mmol/L, and there was wide variation in the individual FFAs.

Geographical variations in the concentration of biliary free fatty acids with anti-mutagenic action

The concentrations and compositions of free fatty acids (FFAs) in human bile, especially of inhibitory free fatty acids (IFFAs), were analyzed in terms of anti-mutagenic effects in relation to the mutagenic activity of bile. Bile samples were collected from patients with cholelithiasis residing in either Niigata or Kochi prefectures of Japan, regions characterized as the highest and lowest risk areas for gallbladder cancer (GBC), respectively. Biliary FFAs and IFFAs were analyzed by high-performance liquid chromatography and mutagenicity was examined in by the Ames test (TA98+S9mix) after blue rayon treatment. There was a tendency for higher biliary FFA and IFFA concentrations in the Kochi subjects, but the proportion of IFFA to the total FFA concentration did not differ between the two areas. There was an inverse correlation between the concentrations of IFFAs and the numbers of revertant colonies in both Niigata and Kochi subjects. However, at a dose of 591 micromol/l, (calculated based on the average amount of IFFAs absorbed in blue rayon) IFFAs did not exhibit anti-mutagenic actions in the blue rayon extracts. Within this range, more positive samples were seen in Niigata than in Kochi, suggesting the presence of more active mutagen(s) in Niigata samples.

Co-mutagenicity of glyco- and tauro-deoxycholic acids in the Ames test.

Mutagenicity and co-mutagenicity of glyco- and tauro-deoxycholic acids (GDCA and TDCA), which are abundant in human bile, were examined by the Ames test. The two chemicals were not mutagenic for themselves to Salmonella typhimurium TA98 and TA100, with and without S9 mix. They enhanced, however, the mutagenic activities of the pro-mutagens, 2-aminoanthracene (2AA) and benzo[a]pyrene (BaP), for both TA98 and TA100 with S9 mix. They were more co-mutagenic for the pro-mutagens on TA98 than on TA100. On TA98, the mutagenic activities of 2AA with GDCA (5 mumol/plate) and with TDCA (5 mumol/plate) were 9.7-fold and 11.8-fold as high as that of the corresponding control (2AA only), respectively. BaP with GDCA (2.5 mumol/plate) and with TDCA (2.5 mumol/plate) showed 2.8-fold and 3.0-fold increases over the corresponding control level (BaP only), respectively. It is hence concluded that GDCA and TDCA may enhance the activity of some mutagens existing in bile.

Mutagenicity of 24-hour duplicate of Japanese diet

In order to elucidate the genotoxicological characteristics of the Japanese diet, the mutagenicity of 24-h duplicate of the diet samples were investigated. The mutagenicity of blue rayon extract was examined in the Ames Salmonella/microsome assay. Thirty-two (91.4%) of 35 samples revealed mutagenicity toward Salmonella typhimurium TA98 in the presence of S9 mix. The mutagenic activities showed significant correlations with the consumption rates of broiled fish (r = 0.517, p = 0.0021) and broiled meat (r = 0.494, p = 0.0036). In other test conditions, 6 (17.1%), 5 (14.3%) and 8 (22.9%) samples were mutagenic to Salmonella typhimurium TA98 without S9 mix, TA100 with S9 mix and TA100 without S9 mix, respectively. Findings in the present study suggest that high consumption of broiled fish and broiled meat are important as the source of mutagens/carcinogens in the Japanese diet. In the present study, however, biological inference of these findings could not be made in relation to the occurrence of cancers, especially of the gastric cancer, which is the most prevalent form of cancer in Japan.