IMPUESTO GENERAL A LAS VENTAS

Even after chromatographic purification, egg and soya PC are heterogeneous molecular mixes (Desai, 1996; section 2.4). Each PC comprises 20 molecular species of PC with different fatty acids. This diversity makes detailed analysis of the molecular breakdown species extremely difficult. The common assay techniques provide an indication of the general oxidative status o f the PL, but do not assess the oxidative condition of the individual molecules.

As a result of the complex processes involved in oxidation, no single test provides an absolute indication of PL stability. A variety of tests were therefore employed in order

Chapter Four-Stability o f unsaturated phospholipids

to detect the different stages of PL oxidation. The tests and assays used in these investigations are described in detail in the following section:

4.3.2.1 Conjugated diene and triene assay

This test was one of the most informative and simple oxidative tests to perform. It involved the detection of conjugated double bonds using UV spectroscopy (Klein, 1970). The measurement of PL absorbance at wavelengths of approximately 230 nm and 275 nm provided an indication of the conjugated diene and triene level respectively. The exact wavelength for the maximum peak varied slightly from lipid to lipid, suggesting that different positioning of the double bonds could alter the precise wavelength at which the diene and triene peaks absorb. A similar observation was also noticed by Memoli et al. (1993).

In order to assess the relative changes in diene and triene levels with time, these absorbances had to be related to the total level o f PL assayed. This was achieved by dividing the absorbance of the conjugated diene or triene by the absorbance of PL at 215 nm. These two ratios are referred to as the conjugated diene oxidation index and the conjugated triene oxidation index respectively.

Sufficient sample containing 5-10 mg of PL was accurately weighed into a 10 ml volumetric flask, dissolved and made up to 10 ml with absolute ethanol. The absorbance of this ethanolic PL solution was recorded over the wavelength 300-200 nm in matched cuvettes against absolute ethanol as the blank using a Perkin Elmer 554 UV spectrophotometer. The wavelengths for the maximum absorbance of the diene and trienes were verified before the absorbance at the wavelengths 215 nm, 230 ± 6 nm, 275 ± 6 nm was measured. To calculate the conjugated diene oxidation index the maximum absorbance of the ethanolic PL at 230 ± 6 nm was divided by the absorbance at 215 nm. Similarly, the conjugated triene oxidation index was calculated by dividing the maximum absorbance at 275 ± 6 nm by the absorbance at 215 nm.

4.3.2.2 Hydroperoxide assay

Traditionally, titrimetric assays are used to measure the hydroperoxide value of PLs. However, in order to detect low levels of peroxidation with the titrimetric assay, large quantities of PL (>6 g) is required for each sample. This quantity of PL was not practical in this study because the three forms were each measured in quadruplicate at five different time intervals. Therefore, an assay which required only milligram

quantities of PL was selected (Takagi et al., 1978). The peroxide assay employed in this study was based on the ability of hydroperoxide to oxidise the iodide ion into an I3. complex. This yellow coloured complex could then be measured spectrophotometrically at 352 nm. The reliability and reproducibility of this assay depended upon: the purging of the assay solvents with nitrogen, sealing of the reaction container with nitrogen to exclude air and the protection of the reaction container from light.

All solutions for this peroxide assay were freshly produced just prior to the assay. The solvents for these solutions were deaerated by bubbling nitrogen through the liquids for 30 minutes. To reduce the evaporation of the volatile solvents during purging, deaeration of these solvents was carried out in containers with narrow necked vessels, whilst being cooled in a bucket of ice.

Instead of assaying hydrated liposomes, the samples were tested for hydroperoxides in their anhydrous forms. This avoided any oxygen dissolved in water interfering with the iodometric assay.

Sufficient sample containing approximately 10 mg of PL was accurately weighed into the bottom of a 5 ml glass bottle. The precise amount depended upon the quality of the PL: highly oxidised material required only a few mg. To this lipid, 1.0 ml of dearated acetic acid: chloroform (3:2 v/v) solution was added and the bottle was sealed under nitrogen. After the PL had fully dissolved, 50 pi of freshly made saturated potassium iodide solution (1.2 g in 1 ml purged deionised water) was added to the PL dissolved in acidified chloroform. The bottle was immediately stoppered, vortexed for 15 seconds and wrapped in foil to protect from light. The sample was placed in the dark for exactly five minutes. After the five minutes had elapsed, 4.0 ml of oxygen free 0.5% w/v cadmium acetate solution was added to the bottle and vortexed for ten seconds. The two phases were separated by centrifugation for 5-10 minutes at 2000 rpm.

Exactly 60 minutes after the addition o f the potassium iodide solution, the absorbance of the upper phase was read at 352 nm, against a blank containing all the components minus PL.

A standard peroxide curve was created using cumene hydroperoxide as the peroxide standard (Appendix I). The cumene hydroperoxide (80%) solution was diluted ten fold by accurately diluting 1.00 ml of the peroxide solution to 10 ml in absolute ethanol (xlO fold dilution). This solution was further diluted by diluting 100 pi of this solution to 10 ml in absolute ethanol (xlOOO fold dilution). The final dilution was made by

Chapter Four-Stability o f unsaturated phospholipids

diluting 1.00 ml of this xlOOO diluted cumene solution to 10 ml in absolute ethanol (final dilution x 10,000 fold). The following amounts of the x 10,000 fold diluted cumene hydroperoxide solution were individually assayed using the peroxide assay: 75 pi, 100 pi, 150 pi, 200 pi, 250 pi. The standard curve was constructed by plotting the amount o f cumene hydroperoxide level (nmol) against the absorption of each cumene hydroperoxide level.

4.3.2 3 Thiobarbituric acid reactive substances (TEARS)

Two thiobarbituric acid (TBA) assays were employed to detect malondialdehyde (MDA). Since MDA is one o f the major breakdown products o f PL degradation, these assays provided an indication of the extent of oxidation. The reaction product of MDA and TBA generated an intense red colour which was detected at a wavelength of 532 nm. These reaction products are widely referred to as thiobarbituric acid reaction substances (TBARS).

The only difference between the two assays used in the stress study was the addition of iron (III) chloride in the TBARS(F) assay prior to heating. The addition of iron chloride to the reaction mixture encouraged the liberation of MDA from hydroperoxides. Hence this variant of the TBARS assay provided an indication of both the hydroperoxide and the secondary product content in the PLs. In contrast, by omitting iron chloride from the reaction mixture, only the secondary products present in the PL were detected (Asakawa and Matsushita, 1979 and 1980).

However, both assays required heating at 95 °C in order for the reactions to proceed. It was therefore necessary to protect the PL from oxidising during the reaction. This was achieved by the addition of the antioxidant butylated hydroxytoluene (BHT) (Asakawa and Matsushita, 1980) to both reaction mixtures prior to heating for 90 minutes at 95 °C.

4.3.2.3.1 Thiobarbituric acid reactive substances with iron (III) chloride (TBARS(F))

A sufficient amount of sample containing about 5 mg o f PL was accurately weighed into a 20 ml glass bottle. To this sample, 100 pi of antioxidant butylated hydroxytoluene solution (110 mg BHT to 10 ml ethanol) was added. After dissolving the PL in this antioxidant solution, 500 pi of deionised water and 100 pi of FeClg solution (27 mg of iron (III) chloride to 10 ml water) were added. This was followed by adding 1.75 ml of

glycine-HCL buffer (0.2 M), pH 3.6. Finally, 1.75 ml o f a solution containing 0.67% w/v thiobarbituric acid and 0.3% w/v sodium lauryl sulphate was added to the glass bottle. The bottle was sealed and vortexed for 15 seconds, before heating for 90 minutes at 95 °C. After heating, the bottles were cooled to room temperature by placing them in water. After cooling, 1.0 ml of acetic acid and 2.0 ml of chloroform were added to the contents o f the bottle and handshaken vigorously. The samples were centriftiged at 5,000 rpm until a visibly clear upper phase was obtained. The absorbance o f the top layer of the sample was measured at 532 nm, against a blank containing all components except for the PL. To ensure the top layer was clear and the absorbance was attributable to pink colouration, the optical density (O.D.) was measured at 560 nm. If the O.D. was low (0.02 and below), this verified that the solution was clear and had been satisfactorily centrifuged.

1,1,3,3- tetraethoxypropane (TEP) was employed as the standard substance for constructing the TBARS curve. This standard substance liberates malondialdehyde and ethanol under mild acid conditions. A standard stock TEP solution was produced by diluting 44 mg TEP to 250 ml with freshly deionised water. The TEP working solution was produced by further diluting 1.0 ml of stock TEP solution to 10 ml with deionised water. The following volumes of TEP working solution were assayed using the TBARS assay omitting the lipid and 500 |il of deionised water: 0 pi , 50 pi, 100 pi, 150 pi, 200 pi, 250 pi, 300 pi and 400 pi. Deionised water was added to the reaction mixture to ensure the volume of the TEP sample totalled 500 pi. A standard curve was constructed by plotting the amount of MDA released (in nmol) against the absorption of each TBARS reacted TEP sample.

4.3.2.3.2 Thiobarbituric acid reactive substances without iron (III) chloride (TBARS)

The method, described in section 4.3.2.4.1, was employed except that iron (III) chloride was omitted from the reaction mixture. The standard curve is not shown, because the curve was identical to TBARS(F) curve (Appendix II).