RESULTS
Diode Array Spectrophotometry
A diode array spectrophotometry was performed in order to ascertain the kinetics of the reaction under varying concentrations of metformin. The following data was collected regarding absorbance versus time measured at 340nm for each trial of the enzyme-coupled reaction, and were graphed using Microsoft Excel 2011. Average absorbances versus time for various tested metformin concentrations against the control are graphed below (see Appendix for raw absorbance versus time graphed for each trial of each concentration group). The slopes of the best fit line for each trial are displayed on the graphs, and demonstrate that the reaction rate for the control was faster than the reaction rates for groups treated with metformin.
A diode array spectrophotometry was performed in order to ascertain the kinetics of the reaction under varying concentrations of metformin. The following data was collected regarding absorbance versus time measured at 340nm for each trial of the enzyme-coupled reaction, and were graphed using Microsoft Excel 2011. Average absorbances versus time for various tested metformin concentrations against the control are graphed below (see Appendix for raw absorbance versus time graphed for each trial of each concentration group). The slopes of the best fit line for each trial are displayed on the graphs, and demonstrate that the reaction rate for the control was faster than the reaction rates for groups treated with metformin.
Figure 5: Average absorbance versus time for 5000µM metformin versus the control are graphed above.
Figure 6: Average absorbance versus time for 2500µM metformin versus the control are graphed above
Figure 7: Average absorbance versus time for 500µM metformin versus the control are graphed above.
Figure 8: Average absorbance versus time for 250µM metformin versus the control are graphed above.
Figure 9: Average absorbance versus time for 1000µM metformin versus the control are graphed above.
Absorbance data was also used to calculate the average net moles of NADH converted into NAD+ after a time interval of approximately 60 seconds in order to quantify the average reaction rate for each concentration group. For each trial, absorbance was multiplied by the corresponding time, and total sums of these products were calculated in order to quantify the net total moles of NADH converted to NAD+ for each trial. These sums were averaged for each concentration group and are graphed below against the control. For all concentration groups except the 5000µM, the net moles of NADH converted into NAD+ was greater for the control than for the experimental group, indicating an overall faster reaction for the control than for most concentration groups of metformin
Figure 10: Average net conversion of NADH into NAD+ for 1000µM against the control is graphed above.
Figure 11: Average net conversion of NADH into NAD+ for 5000µM against the control is graphed above.
Figure 12: Average net conversion of NADH into NAD+ for 2500µM against the control is graphed above.
Figure 13: Average net conversion of NADH into NAD+ for 250µM against the control is graphed above.
Figure 14: Average net conversion of NADH into NAD+ for 500µM against the control is graphed above.
13C-NMR Data
13C-NMRs were performed in order to analyze the presence and number of functional groups, specifically carbonyls, in metformin, oxaloacetate, pyruvate, and biotin, in order to ascertain the interaction between metformin and gluconeogenesis-related compounds. 13C-NMR images for trial 1 of each sample are displayed below (for 13C-NMR images for each trial of the samples, see Appendix).
13C-NMRs were performed in order to analyze the presence and number of functional groups, specifically carbonyls, in metformin, oxaloacetate, pyruvate, and biotin, in order to ascertain the interaction between metformin and gluconeogenesis-related compounds. 13C-NMR images for trial 1 of each sample are displayed below (for 13C-NMR images for each trial of the samples, see Appendix).
Figure 15: Metformin 13C-NMR Trial 1
Figure 16: Biotin 13C-NMR Trial 1
Figure 17: Oxaloacetate 13C-NMR Trial 1
Figure 18: Pyruvate 13C-NMR Trial 1
Figure 19: Metformin-Biotin 13C-NMR Trial 1
Figure 20: Metformin-Oxaloacetate 13C-NMR Trial 1
Figure 21: Metformin-Pyruvate 13C-NMR Trial 1
Statistical Analysis of Absorbance Data
Absorbance values for each trial of the spectrophotometry were inputted into Beer’s Law to find the concentration values of NADH. These values were inputted into an ANOVA using SAS 9.4 software. The ANOVA was performed at a 95% confidence interval, with 𝛼 = 0.05.
Degrees of Freedom - 5
F Value - 2.66
P Value - 0.0473
H0: µi= 0 i = 1, 2, …, 6
H1: at least one of the groups is different, µi ≠ 0
p<𝛼, therefore, the H0 was rejected, and significance in the data set was found.
A Duncan-Waller Post Hoc test was performed in order to ascertain which groups in the data set differed significantly from the control. The following data was collected:
Absorbance values for each trial of the spectrophotometry were inputted into Beer’s Law to find the concentration values of NADH. These values were inputted into an ANOVA using SAS 9.4 software. The ANOVA was performed at a 95% confidence interval, with 𝛼 = 0.05.
Degrees of Freedom - 5
F Value - 2.66
P Value - 0.0473
H0: µi= 0 i = 1, 2, …, 6
H1: at least one of the groups is different, µi ≠ 0
p<𝛼, therefore, the H0 was rejected, and significance in the data set was found.
A Duncan-Waller Post Hoc test was performed in order to ascertain which groups in the data set differed significantly from the control. The following data was collected:
The post hoc test shows that there are significant differences for several groups: control versus 1000µM, control versus 5000µM, and control versus 2500µM.