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The final optimised BHAD enzyme assay is described in Chapter 3 section 3.4.2.3 and was adapted from [13]. BHAD activity is determined by a spectrophotometric assay where conversion of S-acetoacetyl-CoA to ß-hydroxybutyryl-CoA is detected by measurement of absorbance at 340 nm wavelength every 30 seconds for 5 or 10 minutes. The important things for the optimisation of the BHAD assay were a) determination of the amount of muscle protein homogenate to use in the assay and b) the linearity, reproducibility and inter-assay and intra-assay coefficient of variability (CV) of the assay. In order to achieve these aims the standard assay for BHAD was adapted using the rat muscle tissue first and then the spare

human muscle tissue before measuring the BHAD activity in the SPIRIT study participants’

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To determine the amount of rat muscle homogenate to use in the BHAD assay an experiment including increasing volume of muscle protein homogenate (7.0 l to 15 l) was performed. This experiment was repeated three times and Figure 5.13(a) represents results from one experiment. BHAD activity observed with 7 l (0.014 0.002 units/ml, n=3), 10 l (0.015 0.001 units ml, n=3) and 15 l (0.014 0.001 units/ml, n=3) of muscle protein homogenate provided comparable activity. It was decided that 7 l muscle protein homogenate would be used in the final optimised assay (Chapter 3 section 3.4.2.3) because it allowed for more assays to be performed which was very important with very little SPIRIT study human muscle tissue for measurement of enzyme activity.

With the final BHAD assay now optimised using rat muscle homogenate, reproducibility and linearity of the assay (Figure 5.13) as well as examination of the assay in human tissue (Figure 5.14 and Table 5.12) needed to be investigated before embarking on determining BHAD activity in the SPIRIT study participant muscle biopsy sample.

An experiment using the optimised conditions now established for BHAD activity was performed, where BHAD activity was measured in quintuplet using 7 l of the same rat muscle homogenate (Figure 5.13(b)). As can be seen just by visualisation of the activity curves in Figure 5.13(b) the assay is highly reproducible. The intra-assay CV (see Chapter 3 section 3.4.3) for the BHAD assay was 2%. The inter-assay CV (see Chapter 3 section 3.3.3) for the optimised BHAD assay is shown in Table 5.11. The average coefficient of variation between microplate to microplate (n=5) was 3.65%.

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Table 5.11: Inter-Assay Coefficient of Variability for the BHAD assay. Mean of means 0.43

Standard deviation 0.015 % CV of means 3. 65

The inter-assay CV was measured between five microplates (used for the three experiments examining intra-assay CV)

To ensure BHAD activity being measured in the 7 l of muscle protein homogenate is in the linear portion of the BHAD enzyme progress curve, increasing amounts of muscle protein homogenate as shown in Figure 5.13(c) were used to examine BHAD enzyme activity. The 7

l, 10 l and 12 l muscle homogenate exhibited BHAD activity in the linear portion of the BHAD activity curve showing 014 ± 0.01 units/ml (n=3) 0.027 ± 0.0001 units/ ml (n=3) and 0.04 ± 0.0001 units/ ml (n=3) BHAD activity respectively indicating saturation of BHAD enzyme had not been reached as compared to 15 and 17 l of muscle homogenate where the activities of enzyme are as follows) 0.06 ± 0.0001 units/ ml (n=3) and 0.056 ± 0.0001 units/ ml (n=3).

The final optimised BHAD assay needed to be tested in human tissue. The spare human tissue was used to test the assay. Protein homogenate was extracted from the spare human muscle tissue following the optimised protein extraction technique previously described in section 5.3.1. The BHAD assay was performed with increasing amounts of human muscle protein homogenate (5 to 15 l) and 10 l of rat muscle protein homogenate was used as a positive control for the comparison. Figure 5.14 represents results for one experiment and this experiment was done in triplicate. Table 5.12 shows the results for the mean BHAD activity for each homogenate for the three experiments.

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Figure 5.13: Three different experiments measuring BHAD activity to (a) determine

suiTable amount of muscle protein homogenate for the assay (b) examine reproducibility of

the assay and (c) validate linearity of the assay. BHAD activitywas measured in 7, 10 and 15

l of rat muscle homogenate for (a), 7 l of rat muscle homogenate for (b) and 7, 9, 12, 15 and 177 l rat muscle homogenate for (c). BHAD activity was measured at 25 0C and determined spectrophotometrically[13] , with absorbance measured every 30 seconds at 340 nm for 5 minutes.5.4 mM S-acetoacetyl CoA was used as substrate. The blank contained the extraction buffer in the same quantity of muscle homogenate used and all the reagents of the assay.

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Figure 5.14: Comparison of BHAD activity in rat and human muscle protein homogenate. BHAD activity was measured in 10 l of rat muscle homogenate and 7, 10 and 15

g

l human muscle homogenate using 5.4 mM S-acetoacetyl CoA as substrate. BHAD activity was measured at 25 0C and determined spectrophotometrically [13], with absorbance measured every 30 seconds at 340 nm for 10 min. The blank contained the extraction buffer in the same quantity of muscle homogenate used and all the reagents of the assay.

Table 5.12: Beta-hydroxyacyl-CoA dehydrogenase (BHAD) activity in human and rat skeletal muscle

Sample BHAD activity (units/ml) Rat muscle homogenate (10 l) 0.0140 0.0002

Human muscle homogenate

7 l 0.0140 0.0001 10 l 0.0250 0.0001 12 l 0.04 0.0001

15 l 0.06 0.0001 17 l 0.056 0.0001

Data expressed as mean ± SD (n = 3). BHAD activity was measured in 10 l of rat muscle protein homogenate (positive control) and ,7,10, 12, 15, 17 PP l of human muscle protein homogenate. BHAD activity was measured at 25 0_{C and determined spectrophotometrically [13], with absorbance}

measured every 30 seconds at 340 nm for 10 min. 5.4 mM S-acetoacetyl CoA was used as substrate. Units/ml represents mole/min/ml/mg of protein.

0 0.05 0.1 0.15 0.2 0.25 0 200 400 600 absor b ance 340nm Time (seconds) Blank a1= rat tissue a2= human sample a3= human sample a4= human sample

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The 7 l human muscle protein homogenate was the amount used in the final optimised BHAD assay for human muscle tissue. The BHAD activity results for the SPIRT study cohort are presented in section 5.3.5.