Advances in Lipid Methodology. Volume 3
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The effect of UV radiation on lipid peroxidation was monitored. Parts of plants were exposed to UV radiation for days prior the extraction due to induction of lipid peroxidation in plants. UV radiation induced an increase in lipid peroxidation values in all samples of different plant tissues determined by the FOX method. The good correlation was found between the FOX and iodometric methods. However, the iodometric method had limitations in the determination of the low concentrations of lipid hydroperoxides.
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Griffiths et al. They analysed plant tissues, such as extracts of bean hypocotyls Phaseolus sp. Lipid hydroperoxide levels ranged from 26 to nmol. The highest content of lipid hydroperoxides was detected in broccoli and green alga cells in their study. For dilution of stock solutions of standards an epMotion Eppendorf, Germany automated pipetting system was used Fig.
The empty microtubes are placed in the position B3 Fig. Module Reservoir is located in the position B1, where stock solutions are available. The device is controlled by the epMotion control panel. For determination of antioxidant activity, a BS automated spectrophotometer Mindray, China was used. Cuvette content is mixed by an automatic mixer including a stirrer immediately after addition of reagents or samples. Contamination is reduced due to its rinsing system, including rinsing of the dosing needle as well as the stirrer by MilliQ water.
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For detection itself, the following range of wave lengths can be used as , , , , , , , , , , and nm. FOX1 reagents were prepared according Arab et al. The general acidic reagent acidic reagent A final concentrations were 0. The pH of the reagent was adjusted to the value of 1. From the pre-stock solution, five stock solutions: and 0. For further preparation of 20 standards from five stock solutions, an automated pipetting system epMotion was used to minimalize possible pipetting errors. The standards had following concentrations: 0.
These standards were used for the preparation of calibration curves in both manual and automatic measurements. Final value is calculated from the absorbance value of the mixture of the reagent R1 with sample and from the absorbance value after 6 minutes of incubation of the mixture with the reagent R2.
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This mixture was incubated for 4. Final value is calculated from the absorbance value of the mixture of reagent R1 with sample before the addition of the reagent 2 and from the absorbance value after 6 minutes of incubation of the mixture with the reagent 2. Spectrophotometric methods for determination of lipid peroxidation have a relatively simple procedure of a measurement. In addition, they are relatively low-cost with easy applicability and they do not require specialized equipment or personnel.
To maintain the sustainability of these methods, it is necessary to introduce these methods to automated operation, which has not been yet satisfactorily solved. Analyses of samples performed due to intensive work of personnel, which is expensive, slow, and, in addition, the human factor is responsible for a high percentage of errors. Requirement for laboratories, in which a large number of samples is analysed per day, consists in relatively simple and easy to apply method. Our aim was to automate the pre-analytical and analytical phase of the FOX1 method. For specification and comparison of this method, the method based on the use the manual spectrophotometer was also carried out.
Pre-analytical processing of biological samples in the laboratory is a necessary and important part of laboratory work. It represents a wide range of manual, often stereotyped operations that do not require special knowledge and skills, but require maintenance of the standard procedure s and prevent the possibility of errors connected with this analytical phase.
Pre-analytical laboratory process is destined to automation and robotics. Automation and robotics of the pre-analytical phase brings many benefits and advantages to laboratory. It reduces the number of errors, the time necessary for sample manipulation, and the response time. It significantly increases the productivity, cost savings connected with productivity, and minimizes the exposure of personnel with biological material [ ]. For automation of pre-analytical phase, the epMotion automated pipetting system was used. Stock solutions of tert -butylhydroperoxide t -BHP at the concentrations of , , Sixth vial contained diluting solution 0.
Twenty empty Eppendorf tubes 1. Scheme of the preparation of standards is shown in Table 2. Pipetting robot first pipetted different volumes of diluting solution 0. When pipetting the stock solution into the dilution buffer in micro test tube, robot three times mixed the solution by a pipetting. Volume of the solution in the preparation of standards using epMotion automated pipetting system.
Using the epMotion automated pipetting system, work time of 20 minutes was saved time, when laboratory staff was not needed. The only time-demanding operation consisted in replenishment of vials and initiation of the program. Potential errors that arise due to human activity were avoided. Accuracy of a pipetting was verified by weighing, the average error was approximately 1.
Our goal was to introduce the FOX1 method to an automated operation and improve both analysis itself and conditions of analysis. The experiment was carried out using tert -butylhydroperoxide standard prepared at the concentrations from 0. Furthermore, the spectral curves of generated chromatic complexes were observed and the concentration dependence on temperature and time were determined.
In addition, reaction kinetics during the reaction was established. Spectral changes in the t -BHP concentration range from 0. Courses of spectra of t-BHP in the concentrations from 0. C Comparison of values of absorption maximum at the wavelength of nm and a time period of 6 and 60 minutes. All analyses were carried out in triplicates. Absorption maximum at low concentrations up to the concentration of 0. Interaction of sample and reagents proceeded in six minutes, after this time, absorbance could be measured and the final value of lipid peroxidation calculated.
We wanted to determine the changes in the absorbance during one hour. When interlaying the trends points in the linear concentration part from 0. This fact can be explained by unequal reaction kinetics during the analysis see the reaction kinetics, Chapter 3. Dependences of representative concentration On the other hand, this temperature may lead to degradation of biological samples.
Detected at nm, interval of record is 1 minute, interval period 0 - 30 minutes. Automated analyser BS was used for this purpose.
All samples could be studied at all once. This is not possible using the manual spectrophotometer, thus, use the automated analyser represents one of the most important steps in the analysis automation.
The curves were used for the calculating the reaction rate constants indicating the course and conception of the impact of the effect of t-BHP concentration on the reaction rate. The effect of each of concentrations on the change in absorbance value was determined. Monitoring of reaction curves of t-TBH in the concentrations from 0.
Mathematical formularization of the course of reaction curves for t-TBH in the concentration range from 0. Reaction rate constant is expressed as a change in absorbance per second, and per minute. By the using manual spectrophotometer and automated analyser, the dependence of t-TBH concentration 0. The calibration curves were calculated from final values. For other experimental detail, see Fig. A thematic series on rafts is in the works in this journal.
In updating my web pages in the Lipid Essentials section of the LipidWeb, I try to keep the big picture in mind rather than concentrating on the minutiae of a subject. From time to time, there is a substantial change in how a topic is viewed in the lipid community, and I at least have the chance to rectify matters here.
I am thinking now of my web page dealing with rafts , the transient nano-domains reportedly present in cell membranes. This has always been a controversial subject, for example whether "detergent-resistant membranes" are truly representative of rafts, or indeed whether rafts do indeed exist. When I first prepared a web page on this subject, it was based entirely on the premise that there was a specific interaction between sphingolipids, especially sphingomyelin, and cholesterol in membranes that determined the existence and function of rafts.