EPR spin trapping detection of carbon-centered carotenoid and beta-ionone radicals

Free radical intermediates were detected by the electron paramagnetic resonance spin trapping technique upon protonation/deprotonation reactions of carotenoid and beta-ionone radical ions. The hyperfine coupling constants of their spin adducts obtained by spectral simulation indicate that carbon-centered radicals were trapped. The formation of these species was shown to be a result of chemical oxidation of neutral compounds by Fe(3+) or I(2) followed by deprotonation of the corresponding radical cations or addition of nucleophilic agents to them. Bulk electrolysis reduction of beta-ionone and carotenoids also leads to the formation of free radicals via protonation of the radical anions. Two different spin adducts were detected in the reaction of carotenoid polyenes with piperidine in the presence of 2-methyl-2-nitroso-propane (MNP). One is attributable to piperidine radicals (C(5)H(10)N*) trapped by MNP and the other was identified as trapped neutral carotenoid (beta-ionone) radical produced via protonation of the radical anion. Formation of these radical anions was confirmed by ultraviolet-visible spectroscopy. It was found that the ability of carotenoid radical anions/cations to produce neutral radicals via protonation/deprotonation is more pronounced for unsymmetrical carotenoids with terminal electron-withdrawing groups. This effect was confirmed by the radical cation deprotonation energy (H(D)) estimated by semiempirical calculations. The results indicate that the ability of carotenoid radical cations to deprotonate decreases in the sequence: beta-ionone > unsymmetrical carotenoids > symmetrical carotenoids. The minimum H(D) values were obtained for proton abstraction from the C(4) atom and the C(5)-methyl group of the cyclohexene ring. It was assumed that deprotonation reaction occurs preferentially at these positions.     

The preservation of salt marsh algae by controlled liquid nitrogen freezing.

Five species of benthic marine algae were preserved by controlled liquid nitrogen freezing and storage over periods extending to 1 year. Only a small percent of the algae survived without cryoprotectant. Nannochloris adamsii was an exception; 67% survived after 12 months of storage. Nitzschia acicularis was the best preserved with 5 glycerol as a cryoprotectant, Dimethylsulfoxide was a better cryoprotectant for N. adamsii and Dunaliella quartolecta. Reducing normal brackish salinity (28‰) of the culture medium to one half (14‰) increased the survival percentages for N. acicularis, Cylindrotheca closterium and Phaeodactylum tricornutum. The morphology and physiology of the species tested were unchanged by long storage time in liquid nitrogen.     

Growth kinetics of cells following freezing in liquid nitrogen

The growth kinetics of cells frozen to ?196 °C were monitored after thawing by various techniques. Progression through the cell cycle in the exposed generation was observed by monitoring cell growth either via multiplicity counts or by electronic cell counts of trypsinized suspensions. Subsequent generations were followed by time-lapse microcinematography.The division delay in the exposed generation of exponential-phase cells was dependent on cell age at the time of freezing and varied from 4 to 8 hr. The time of the first generation was still prolonged significantly but subsequent generations revealed cell cycle times that are comparable to unfrozen cells. In the case of plateau-phase cells, mitosis was delayed 7 hr in the exposed generation. This is 50% longer than the delay seen for pre-DNA synthetic g₁ cells in exponentially growing cultures.A rather important observation in this study was that frozen-thawed cells which divide once will probably continue dividing whereas eventual nonsurvivors are not likely to divide at all. The latter, however, remain active for more than 35 hr as observed microscopically, hence possibly indicating residual metabolic activity.     

Ultrastructural preservation of plasma membranes by non-lethal slow freezing to liquid nitrogen temperature

Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196 degrees C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196 degrees C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.     

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen

Survival of Nippostrongylus brasiliensis larvae after freezing over liquid nitrogen. International Journal for Parasitology4: 173–176. Third stage larvae of N. brasiliensis were frozen over liquid nitrogen and after storage for 7 days were thawed rapidly and inoculated subcutaneously into rats. When ensheathed larvae were used, none survived freezing as judged by motility and infectivity trials. Separate vials of exsheathed larvae survived freezing in proportions ranging from 10 to 64 per cent. Female worms, derived from frozen exsheathed larvae, had a normal complement of eggs in the uterus and both male and female worms had a normal histological appearance. Exsheathed larvae frozen in the presence of 10 per cent dimethylsulphoxide had the same survival rate as those frozen without the addition of cryoprotectant. The addition of 10 per cent glycerol adversely effected the survival of frozen exsheathed larvae.     

The freezing and preservation of bovine erythrocytes in liquid nitrogen

Bovine erythrocytes can be preserved for long periods of time by freezing and storing in liquid nitrogen. Blood group determinations, titrations, and isoimmunizations indicated that there were no detectable alterations in antigenic reactivities of preserved erythrocytes when compared with fresh samples of blood from the same animal. In addition, consistent percentages of recovery within a frozen mixture could be obtained, indicating that the deterioration of erythrocytes with time was not significant. Variations in the percentage of recovery of erythrocytes with different concentrations of sucrose (the cryoprotective agent) were significant at the 0.01 level of probability. The highest average percentages of recovery of erythrocytes from whole blood and the cellular fraction of blood (blood from which plasma was removed before freezing) were obtained with 45 and 40% sucrose, respectively. Results also indicated that whole blood gave a slightly higher percentage of recovery, than the cellular fraction. The individual donor effect, storage time in liquid nitrogen, and various saline concentrations of the thawing and washing solutions had no significant effects upon percentage of recovery.     

A new approach for EPR detection of hydroxyl radicals by reaction with sterically hindered cyclic amines and oxygen

Sterically hindered cyclic amines react with hydroxyl radicals in the presence of oxygen to yield stable nitroxide radicals which can be detected by EPR. This reaction provides a nonconventional spin-trapping tool for detection of hydroxyl radicals.     

Improved method for the cryopreservation of human red cells in liquid nitrogen with hydroxyethyl starch.

Refinement of a method described previously (Cryobiology12, 110–118, April, 1975) made possible routine freezing of full units of packed erythrocytes after separation of platelet rich plasma, and buffy coats. The volume frozen was 405 ml which included packed red cells (190–220 ml), plasma (43–73 ml), and cryo-HES (142 ml, final concentration 14% $\text{w}\text{v}$). The units could be frozen with or without shaking by direct immersion in liquid nitrogen. Thawing was accomplished by transferring units quickly from liquid nitrogen storage to a shaking water bath at 54 °C. The average yield from units of red cells was 98.4%. The stability to a 50-fold dilution in 0.15 m NaCl was 87.8%. Thawing rate was the critical variable in producing the most stable thawed cells. Plasma expander HES was usable but the thawed units were more viscous and about 7% less stable. Red cells prewashed with 0.15 m NaCl and frozen without plasma showed no significant changes in cellular yield or stability. The optimum resuspension medium was 3% glucose. A morphologic study of cells fixed in 1% glutaraldehyde revealed that before freezing red cells were partially dehydrated in 14% HES. These were smooth, flat discs. Cells fixed on thawing were extensively dehydrated and seen as large, thin, smooth, flat discs with approximately 10% echinocytes. On dilution with 6% glucose (1:1) these swelled and reverted to biconcave discocytes except for approximately 5% echinocytes. Storage in liquid nitrogen measured in groups of three units of 15 units for 0, 3, 6, 9, and 12 weeks revealed normal postthawed oxygen delivery (P-50). The greatest measurable effect of freezing red cells in HES was a loss of cellular K⁺ compensated by a corresponding increase in Na⁺.     

EPR spin trapping of protein radicals

Electron paramagnetic resonance (EPR) spin trapping was originally developed to aid the detection of low-molecular-mass radicals formed in chemical systems. It has subsequently found widespread use in biology and medicine for the direct detection of radical species formed during oxidative stress and via enzymatic reactions. Over the last 15 years this technique has also found increasing use in detecting and identifying radicals formed on biological macromolecules as a result of either radical reactions or enzymatic processes. Though the EPR signals that result from the trapping of large, slowly tumbling radicals are often broad and relatively poor in distinctive features, a number of techniques have been developed that allow a wealth of information to be obtained about the nature, site, and reactions of such radicals. This article summarizes recent developments in this area and reviews selected examples of radical formation on proteins.     

植物挥发性物质提取的一种简易方法--液氮冷凝法

介绍了植物挥发物提取的一种简易方法——液氮冷凝法。利用液氮的制冷原理对植物挥发性物质进行提取收集,当携带植物挥发性物质的纯净空气带动供试植物组织的挥发性物质通过一置有U形玻璃管的液氮罐时,由于液氮的低温作用,挥发性物质在U形管内壁上遇冷凝结。该提取装置简单,由硅胶柱、活性碳柱、流量计、锥形瓶、U形管、液罐及各种连接管构成,容易组装。利用该装置提取的挥发性物质可以用于生测,去除水分后还可用于电生理以及挥发性物质化学组成分析等。     

Simplified method for monitoring tricyclic antidepressant therapy using gas—liquid chromatography with nitrogen detection

A simplified gas chromatographic method for the rapid measurement of tricyclic anti-depressant drugs in plasma using a nitrogen-sensitive detector is described. All drugs are extracted and chromatographed under identical conditions. Tertiary amines are separated from their secondary amine metabolites, which are determined simultaneously without the need for derivatisation. The lower limit of accurate determination for most drugs is 10 μg/1.

The method has been applied to the routine measurement of amitriptyline and nortriptyline in plasma from patients receiving antidepressant treatment. Large and important interindividual differences in plasma concentrations in the patients investigated have been found, and the significance of these results is discussed.