Lipase-Catalyzed Synthesis of Unsaturated Acyl L-Ascorbates and Their Ability to Suppress the Autoxidation of Polyunsaturated Fatty Acids

ABSTRACT: L-Ascorbic acid and various polyunsaturated fatty acids (PUFA) were condensed at 55°C by the immobilized lipase Chirazyme L-2 in dry acetone to produce the unsaturated acyl ascorbates. The PUFA moieties of the products were much more resistant to autoxidation at 65°C and nearly 0% relative humidity than the corresponding unmodified PUFA. The effects of the molar ratio of ascorbic acid or linoleoyl ascorbate to linoleic acid on the autoxidation of linoleic acid were examined. The autoxidation of linoleic acid was effectively suppressed at molar ratios greater than or equal to 0.2 when either ascorbic acid or linoleoyl ascorbate was mixed with linoleic acid. The addition of lauroyl ascorbate, synthesized through the enzyme-catalyzed condensation of ascorbic acid and lauric acid in acetone, to docosahexaenoic acid also significantly suppressed the autoxidation of docosahexaenoic acid at molar ratios of ≥0.2. Paper no. J9826 in JAOCS 78, 823-826 (August 2001). KEY WORDS: Acyl ascorbate, L-ascorbic acid, autoxidation, condensation, immobilized lipase, polyunsaturated fatty acid. Much attention has been paid to the use of polyunsaturated fatty acids (PUFA) as components in foods (1). However, PUFA are susceptible to autoxidation (2,3), and the autoxidation causes deterioration of the foods. L-Ascorbic acid is a hydrophilic antioxidant with a strong reducing ability. The lipase-catalyzed synthesis of acyl ascorbate in a solid-phase system (4) or in an organic solvent (5-9) has been reported. However, its ability to suppress lipid autoxidation has not been reported. In a previous paper (10), we reported the synthesis of 6-O-eicosapentaenoyl L-ascorbate by the lipase-catalyzed condensation of eicosapentaenoic acid and L-ascorbic acid in acetone and compared its autoxidation process to that of the unmodified eicosapentaenoic acid. In the work described in this paper, some PUFA L-ascorbates were synthesized using an immobilized lipase from Candida antarctica, Chirazyme ® L-2, and their autoxidation processes were then observed. The PUFA used were linoleic, α-linolenic, γ-linolenic, arachidonic, and docosahexaenoic acids. The effect of the molar ratio of unmodified L-ascorbic acid or linoleoyl ascorbate to linoleic acid on the suppression of the autoxidation of linoleic acid was examined. We previously reported the lipase-catalyzed condensation of ascorbic acid with various medium-chain fatty acids having carbon numbers of 6, 8, 10, and 12 in acetonitrile (11). Therefore, the ability of lauroyl ascorbate to suppress the autoxidation of docosahexaenoic acid was also evaluated in the present work. EXPERIMENTAL PROCEDURES Materials. γ-Linolenic and docosahexaenoic acids were supplied by the Maruha Corporation (Tokyo, Japan), and their purities were both greater than 95% based on gas chromatographic (GC) analysis. L-Ascorbic acid, linoleic acid, acetone, and hexane were purchased from Nacalai Tesque (Kyoto, Japan). α-Linolenic, arachidonic, and lauric acids were purchased from Sigma Chemical (St. Louis, MO). Immobilized lipase from C. antarctica, Chirazyme ® L-2 c.-f. C2, was obtained from Roche Molecular Biochemicals (Mannheim, Germany). The enzyme is the same as Novozym ® 435 according to the manufacturer. Soybean oil was purchased from Wako Pure Chemical Industries (Osaka, Japan). Condensation reaction. Acetone was first dehydrated by adding 5 Å molecular sieves. The water content of the acetone was about 0.01% (vol/vol), and was determined for each experiment by a Karl-Fischer titration. L-Ascorbic acid (0.125 mmol) and a PUFA [linoleic acid (0.577 mmol)], γ-linolenic acid (0.600 mmol), arachidonic acid, (0.638 mmol), α-linolenic acid (0.611 mmol), and docosahexaenoic acid (0.648 mmol)] were weighed into an amber glass vial with a screw cap, and 200 mg of Chirazyme L-2 and 2.5 mL of dehydrated acetone were added to the vial. The headspace of the vial was filled with nitrogen, and the vial was tightly sealed. The vial was then immersed in a waterbath at 55°C with vigorous shaking to commence the condensation reaction. At appropriate intervals, 10 µL of the reaction mixture was taken and mixed with 50 µL of a 50 mM solution of toluene in high-performance liquid chromatography (HPLC) eluent [acetonitrile/tetrahydrofuran/0.1% (vol/vol) phosphoric acid (50:22:28 by vol) as the internal standard for the HPLC analysis and then with 40 µL of HPLC eluent. The analysis was carried out by HPLC with a YMC-Pack C8 column (4.6 mm × 250 mm; YMC Inc., Kyoto, Japan) and an ultraviolet (UV) detector (245 nm). The mixture (20 µL) was applied to the column and eluted with the eluent at 1.5 mL/min. The retention times o

Patterns of C-reactive protein trends during clozapine titration and the onset of clozapine-induced inflammation: a case series of weekly and daily C-reactive protein monitoring

BackgroundInternational guidelines for clozapine titration recommend measuring C-reactive protein (CRP) weekly for 4 weeks after clozapine initiation to prevent fatal inflammatory adverse events, including myocarditis. However, limited evidence exists regarding whether weekly CRP monitoring can prevent clozapine-induced inflammation.AimsWe examined the relationship between CRP trends and the development of clozapine-induced inflammation. We also explored the usefulness and limitations of CRP monitoring during clozapine titration.MethodThis study presents 17 and 4 cases of weekly and daily CRP monitoring during clozapine initiation, respectively.ResultsAmong 17 patients with weekly CRP measurements, 7 had fever. Elevated CRP levels were detected before the onset of fever in two of the seven patients. Of the five remaining patients, the CRP levels on a previous test had been low; however, the fever developed suddenly. Of the 10 patients with no fever under weekly CRP monitoring, three had elevated CRP levels >3.0 mg/dL. Refraining from increasing the clozapine dose may have prevented fever in these patients. Among four patients with daily CRP measurements, two became febrile. In both cases, CRP levels increased almost simultaneously with the onset of fever.ConclusionWeekly and daily CRP monitoring during clozapine titration is valuable for preventing clozapine-induced inflammation, assessing its severity, and guiding clozapine dose adjustments. Weekly CRP monitoring may not adequately predict clozapine-induced inflammation in some cases. Consequently, clinicians should be aware of the sudden onset of clozapine-induced inflammation, even if CRP levels are low. Daily CRP monitoring is better for detecting clozapine-induced inflammation

The need for transparency and good practices in the qPCR literature.

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not