TALLER N° 2 La autoestima

3.3.1 Muscle sample preparation

The analysis of taurine content required that the muscle samples be freeze-dried prior to the taurine extraction process. As the typical dry weight to wet weight ratio is 1:4, at least 10-15 mg of muscle was required to ensure sufficient freeze-dried tissue was available for extraction. Prepared muscles were weighed into cryules with vented lids, and placed in a pre-cooled freeze drier (-40 °C) (Edwards Modulyo, Edwards High Vacuum, Britain, England) for a minimum of 48 hours.

After 48 hours muscle samples were removed from the freeze-drier and placed in a desiccator at room temperature for a minimum of 1 hour. Samples were then weighed to determine the dry to wet weight percentage (% dw/ww), as this is a measure of the effectiveness of the freeze drying process and water content of the sample. After the % dw/ww was determined, samples were crushed to powder with a mortar and pestle and then observed under a microscope, so any blood or connective tissue could be removed. The crushing process was kept to a maximum of 10 minutes, to ensure that the sample did not regain excessive moisture. Two milligrams of powdered muscle was then weighed out into an eppendorf tube and stored in a desiccator until all samples were ready for the taurine extraction process. All samples were crushed and extracted on the day of removal from the freeze drier.

3.3.2 Taurine extraction

Taurine extraction was performed on ice with all extraction solutions and tubes pre- cooled to 0 °C. The tube containing 2 mg of powdered muscle was removed from the desiccator and 20 % w/v of sulfosalicylic acid was added to the tube, before the sample was vortexed for 5 seconds and then placed on ice. This process was then repeated for the remaining samples, with no more than 12 samples being extracted at one time. Once all the samples to be extracted were on ice, each sample was vortexed for a further 5 seconds before the tissue was tapped down to the bottom of the tube and the sample placed back into the ice, with this process being repeated for a total of 10 cycles. At the 10 minute mark, the samples were transferred to a pre-cooled centrifuge (0 °C)

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and spun at 28,000 RPM for 2 minutes. The samples were then removed from the centrifuge and a 125 µl aliquot of supernatant was carefully isolated without disturbing the pellet, and placed into another pre-cooled tube. To neutralise the sulfosalicylic acid in the extract, 0.4 M Borate buffer was added, and the sample then vortexed for 10 seconds before being placed back on ice for 5 minutes. After 5 minutes, the samples were vortexed again, and then centrifuged as before at 28,000 RPM, 0 °C for 2 minutes. The supernatant (taurine extract) was removed using a pasteur pipette and placed in a labelled cryule, before being stored at -80 °C until further processing for the measurement of taurine content.

3.3.3 Measurement of taurine content

The most common method of measuring taurine content reported in the literature is via HPLC, using taurine extracts that have been derivatised with o-phthalaldehyde. Our lab has previously used this method however; several difficulties were experienced with our HPLC setup resulting in variable data when samples were not all analysed within the same run. Due to the large number of samples needing to be analysed for this thesis, this method was insufficient for taurine analysis in this case. Thus, in consultation with a

specialist HPLC analysis company, the method of derivatisation with

fluorenylmethyloxycarbonyl (FMOC) was employed, as it achieves extremely accurate and reproducible measures of taurine content and improves stability of the sample.

The FMOC derivatisation cocktail consisted of 0.4 mM of FMOC in Acetonitrile, 0.2 M NaHCO3 and millli-Q water, which were mixed into a 5 ml screw cap vial on the day of

extraction. Taurine extract (100 µl) was then added to the vial containing derivatisation cocktail, and the solution vortexed vigorously for 30 seconds. The solution was then left to stand for 30 minutes at room temperature (20-23 °C). Spiked samples (samples with a known concentration of taurine standard added) were prepared in the same way, however 10 µl less milli-Q water was added to allow for the addition of 10 µl of taurine standard. After 30 minutes, pentane was added to the vial and then shaken well, to ensure that any unreacted FMOC was removed. The vial was then left to allow the layers of solution to separate (pentane on top, derivatised solution on the bottom). An aliquot of the derivatised solution was then removed using a pasteur pipette, ensuring that no pentane was disturbed, and was placed in a 1 ml HPLC vial which remained

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uncapped for 1 minute to allow for any pentane that may have been transferred with the derivatised sample to evaporate. The vial was then capped and stored at 4 °C until delivery to ACS laboratories for HPLC analysis.

Figure 3.2 Typical HPLC trace for taurine content analysis

Figure shows taurine content as an FMOC derivative in a typical sample, a standard and a blank.

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