When larvae of the salamander Hynobius retardatus were reared at a high temperature (28°C) during their thermosensitive period (TSP: from 15 to 30 days after hatching), all larvae developed to phenotypic females irrespective of their genetic sexes. Hynobius P450 aromatase (P450arom) and Dmrt-1 cDNAs were isolated and their expression pattern was analyzed by competitive and conventional RT-PCR. While P450arom gene was expressed predominantly in the ovary, Dmrt-1 was expressed exclusively in the testis. When larvae were reared at the female-producing temperature (28°C) during the TSP, strong expression of the P450arom and complete suppression of the Dmrt-1 genes were induced in all experimental larvae. Up-regulation of the P450arom and down-regulation of Dmrt-1 genes even in genetic males constitute a part of the molecular biological cascade for the temperature-dependent sex reversal from genetic males to phenotypic females in this salamander.
Recent concerns about potential health risks associated with 3-chloro-1,2-propanediol fatty acid esters (3-MCPD esters) and glycidyl fatty acid esters (GEs) in refined vegetable oils and fats have led to development of various methods for quantitative determinations of these compounds. Among them, indirect methods, which determine total amount of 3-MCPD and glycidol after hydrolysis of the esters, have an advantage over direct methods, which require a reference standard for each of the many possible species of fatty acid esters to be quantified. The existing indirect methods, however, may yield unreliable results or require long hours of alkaline methanolysis. To overcome these shortcomings, the objective of this study was to develop a reliable and rapid indirect method for simultaneous determinations of 3-MCPD esters and GEs. By using Candida rugosa lipase and sodium bromide for a hydrolysis/bromination step, 3-MCPD esters and GEs were transformed within 30 min to 3-MCPD and 3-bromo-1,2propanediol (3-MBPD), respectively, without interconversion. Gas chromatography-mass spectrometry (GC-MS) analysis of 3-MCPD and 3-MBPD derivatized with phenylboronic acid showed 89-108 % recovery of 3-MCPDdiesters, -monoesters, and GEs from various oils and fats spiked at 2 and 20 mg/kg. The method should be useful for routine analysis of 3-MCPD esters and GEs.Keywords 3-MCPD fatty acid ester Á Glycidyl fatty acid ester Á Simultaneous determination Á Indirect method Á Lipase
A collaborative study was conducted to evaluate an indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. The method is characterized by the use of Candida rugosa lipase, which hydrolyzes the esters at room temperature in 30 min. Hydrolysis and bromination steps convert esters of 3-MCPD, 2-MCPD, and glycidol to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol, respectively, which are then derivatized with phenylboronic acid, and analyzed by gas chromatographymass spectrometry. In a collaborative study involving 13 laboratories, liquid palm, solid palm, rapeseed, and rice bran oils spiked with 0.5-4.4 mg/kg of esters of 3-MCPD, 2-MCPD, and Gly were analyzed in duplicate. The repeatability (RSD r ) were < 5% for five liquid oil samples and 8% for a solid oil sample. The reproducibility (RSD R ) ranged from 5% to 18% for all oil samples. These RSD R values were considered satisfactory because the Horwitz ratios were ≤ 1.3% for all three analytes in all oil samples. This method is applicable to the quantification of 3-MCPD, 2-MCPD, and Gly esters in edible oils.
Because of the potential health risks, fatty acid esters of 3‐chloro‐1,2‐propanediol (3‐MCPD‐Es), 2‐chloro‐1,3‐propanediol (2‐MCPD‐Es) and glycidol (Gly‐Es) in foods are drawing the attention of public health authorities. To assess applicability of the rapid indirect method developed earlier by using a Candida rugosa lipase for the analyses of refined fats and oils was applied to the analyses of various foods. Mayonnaise, vegetable oil margarine and fat spread could be analyzed with the hydrolysis condition of 30 min at room temperature. Analyses of 3‐MCPD‐Es in margarines and fat spreads containing milk fat could be analyzed by increasing the hydrolysis temperature to 40 °C. The results in a mayonnaise, four fat spreads and five margarines analyzed by the enzymatic method were 0.10–0.98 mg/kg for 3‐MCPD, 0.05–0.41 mg/kg for 2‐MCPD and 0.15–0.59 mg/kg for Gly, and correlated well with the results obtained by AOCS Cd 29a with Cd 30–15 with slopes of 0.99–1.13, and R2s of 0.87–0.99. Further, by adding a simple fat extraction step using a solvent mix at 60 °C, foods high in protein and carbohydrate, such as infant formulas, could also be successfully analyzed with >90 % recovery in 1 day. Because the enzymatic method requires only 30 min for hydrolysis, the method is considered suitable for routine analyses of 2‐/3‐MCPD‐Es and Gly‐Es in foods.
We developed a novel, indirect enzymatic method for the analysis of fatty acid esters of 3-monochloro-1,2-propanediol (3-MCPD), 2-monochloro-1,3-propanediol (2-MCPD), and glycidol (Gly) in edible oils and fats. Using this method, the ester analytes were rapidly cleavaged by Candida rugosa lipase at room temperature for 0.5 h. As a result of the simultaneous hydrolysis and bromination steps, 3-MCPD esters, 2-MCPD esters, and glycidyl esters were converted to free 3-MCPD, 2-MCPD, and 3-monobromo-1,2-propanediol (3-MBPD), respectively. After the addition of internal standards, the mixtures were washed with hexane, derivatized with phenylboronic acid, and analyzed by gas chromatography-mass spectrometer (GC-MS). The analytical method was evaluated in preliminary and feasibility studies performed by 13 laboratories. The preliminary study from 4 laboratories showed the reproducibility (RSD R ) of < 10% and recoveries in the range of 102-111% for the spiked 3-MCPD and 2-MCPD in extra virgin olive (EVO) oil, semi-solid palm oil, and solid palm oil. However, the RSD R and recoveries of Gly in the palm oil samples were not satisfactory. The Gly content of refrigerated palm oil samples decreased whereas the samples at room temperature were stable for three months, and this may be due to the depletion of Gly during cold storage. The feasibility studies performed by all 13 laboratories were conducted based on modifications of the shaking conditions for ester cleavage, the conditions of Gly bromination, and the removal of gel formed by residual lipase. Satisfactory RSD R were obtained for EVO oil samples spiked with standard esters (4.4% for 3-MCPD, 11.2% for 2-MCPD, and 6.6% for Gly).
Expert Committee on Food Additives (JECFA) evaluated 3-MCPDEs, 3-MCPD, GEs and glycidol and recommended that efforts to reduce 3-MCPDEs and 3-MCPD in infant formula be implemented and that measures to reduce GEs and glycidol in fats and oils continue, particularly when used in infant formula 9) . In 2018, the European Commission started the enforcement of maximum levels for GEs in edible vegetable fats and oils, infant formula, follow-on formula, and foods for special medical purposes intended for infants and young children 10) .Concerns over the potential health risks of orally ingested 3-MCPDEs and GEs have prompted the development of quantitative methods for these compounds in edible fats Abstract: For indirect determination of 3-chloro-1,2-propanediol fatty acid esters (3-MCPDEs) and glycidyl fatty acid esters (GEs) in thermally processed foodstuffs distributed in Japan, we modified two published methods, an enzymatic method (later approved as JOCS Standard Method for the Analysis of Fats, Oils, and Related Materials 2.4.14 -2016 and Joint JOCS/AOCS Official Method Cd 29d-19) and EFSA method developed by the Joint Research Centre of the European Commission. The performance of these methods was demonstrated to be satisfactory. The partially modified enzymatic method showed mean recoveries of 93.7-98.5% for 3-MCPDEs, 94.4-98.4% for GEs, and HorRat (r) values of 0.06-0.78 in analyses of 6 types of foods including Japanese specific foods (fried rice cracker, fried instant noodle, biscuit, karinto, vegetable tempura, and frozen deep-fried chicken) spiked with 3-MCPD dioleate and glycidyl oleate at 0.02-0.04 mg/ kg or 0.2-0.4 mg/kg. The partially modified EFSA method showed mean recoveries of 96.6-99.4% for 3-MCPDEs, 95.7-100.1% for GEs, and HorRat (r) values of 0.14-1.05 in analyses of 5 types of foods (not including karinto) spiked simultaneously with 3-MCPD dioleate and glycidyl oleate at either 0.02-0.04 mg/ kg or 0.2-0.4 mg/kg. The results of analyses of 9 samples (fried rice cracker, biscuit, 2 potato crisps, fried potato snack, baked cracker, cracker dough, seafood tempura, and frozen deep-fried chicken) using these 2 methods were comparable. The 95% confidence intervals determined with weighted Deming regression analysis between the results of 3-MCPDEs or GEs in the same samples analyzed by the 2 methods showed: the slope around 1 (3-MCPDEs, 0.982-1.025; GEs, 0.887-1.078); and intercept close to 0 (3-MCPDEs, -0.002-0.003; GEs, -0.011-0.015). These data confirmed that the concentrations of 3-MCPDEs and GEs in food samples determined by 2 independent analytical methods were equivalent.
An indirect enzymatic analysis method for the quantification of fatty acid esters of 2-/3-monochloro-1,2-propanediol (2/3-MCPD) and glycidol was developed, using the deuterated internal standard of each free-form component. A statistical method for calibration and quantification of 2-MCPD-d, which is difficult to obtain, is substituted by 3-MCPD-d used for calculation of 3-MCPD. Using data from a previous collaborative study, the current method for the determination of 2-MCPD content using 2-MCPD-d was compared to three alternative new methods using 3-MCPD-d. The regression analysis showed that the alternative methods were unbiased compared to the current method. The relative standard deviation (RSD) among the testing laboratories was ≤ 15% and the Horwitz ratio was ≤ 1.0, a satisfactory value.
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