Effect of Inflammation on lipid nanoparticle performance

The purpose of this study is to determine the inhibition constant (Ki) of beta glycerol phosphate as an inhibitor of alkaline phosphatase using 4-Methylumbelliferyl phosphate (4-MUP) fluorescent dye. Alkaline phosphatase (ALP) is an enzyme responsible for the hydrolysis of organic phosphate esters present in extracellular space. In other words, it removes phosphate groups from other molecules, such as nucleotides and proteins. The tissue-nonspecific isozyme of ALP (TNAP) is commonly found in bone. When the ALP enzyme hydrolyzes the pyrophosphates and phosphate esters present, inorganic phosphate is supplied to the bone tissue, enhancing mineralization. Beta glycerol phosphate is a substrate of ALP, typically found in the liver. ALP activity can be visualized with many different fluorescent techniques, such as using 4-Methylumbelliferyl phosphate dye. An ALP stock solution of 20 U/mL was used, as were stock solutions of 200 mM and 10 mM for beta-GP and 4-MUP, respectively. These solutions were serially diluted into working solutions. The working solutions consisted of a 0.1 U/mL of ALP, which would be used for each well containing ALP in the 96 well plate, as well as 0.05 mM, 0.1 mM, 0.5 mM, 1mM, and 2 mM for beta-GP, and 20 µM ,50 µM, 100 µM, and 200 µM for 4-MUP, which would be varied in wells according to the plate layout. By conducting a plate reading, the fluorescent dye was able to detect enzyme activity when different concentrations of the beta-GP inhibitor were added. A trend showing that an increase in beta-GP concentration was directly related to a decrease in ALP activity was observed, confirming that beta-GP does act as inhibitor to ALP. The inhibition constant (Ki) found was 240.29 µM. This value is like that found in related literature. ALP activity was also found to be higher at pH 9.25 than at neutral pH.

Hello, my name is Peter Touma and my project is using beta glycerol phosphate as an inhibitor of alkaline phosphatase. The purpose of this study was to determine the inhibition constant or Ki of beta glycerol phosphate as an inhibitor of ALP using the 4-Mup fluorescent dye at an alkaline and neutral PH. This is being done to see if beta GP can act as an inhibitor of a op at physiological Ph as well as to see how great the inhibition is when different concentrations of beta GP are used. ALP enzymes are found naturally in the bloodstream liver bone and the kidney ALP is also responsible for the hydrolysis of organic phosphate ethers present in the extracellular space. And beta GP is a subtrate found in bone tissues and promotes mineralization. In blood coagulation. Beta GP could inhibit ALP activity towards other organic phosphates as well. The hypothesis of this study was that beta GP would act as a competitive inhibitor with the form EP substrate for calf intestinal alkaline phosphatase. Solutions were placed accordingly to the plate layout with column six and seven containing the positive and negative controls respectively. The same plate layout was used for each PH tested. Stock solutions were made for the A LP. Beta GP and four Mup. And then serial dilutions were made using these and placed in the 96 world plate accordingly. Stock solution to ALP was 20 units per milliliter. While for four Mup it was 10 milli molar and Beta GP had a 200 milli molar stock solution solutions included a 0.4 unit per milliliter working solution. A LP which had a final concentration of 0.1 units per milliliter in the plate. Beta GP had working solutions of 0.1 milli molar, 0.512 and 0.05 milli molar as well. Or MEP had working solutions of 20 micro molar, 50 micro molar, 100 micro molar and 200 micro molar LB plots are made for each concentration of beed GP that was used using these plots. The KM could be found by obtaining the X intercept. The Km found in each line bid plot were used to generate a Dixon plot. This was done by dividing the CAM found which is known as the cam apparent by the true Km which is the KM when there is no inhibitor added the Dixon plot generated can then be used to find the K I of beta GP. By taking the inverse of the slope. Mila’s mentum plots show the relationship between the concentration of a substrate and the rate of which enzyme reaction is taking place, he remade Mila’s mentum plots at ph seven and 9.25. We see that there’s a clear increase in innovation with an increase in beta GP concentration in both of these plots at both Ph’s. ALP activity as expected was also higher at ph 9.25 compared to neutral PH. But the enzyme activity was still present in neutral Ph and could be inhibited by beta GP in dose dependent manners. The K I found in the study was 2 40 micro molar. This is very similar to the K I found in literature where it was 250 micro molar. We used the PH of 8.39 0.25 and seven. In our study, the K I was not obtained at neutral and alkaline ph. But the A OP activity was seen to be higher alkaline Ph than at neutral Ph beta GP was also shown to have a large inhibitory effect on ALP. And higher concentrations of beta GP did show to have larger inhibition as the change in initial velocity was larger at lower concentrations of beta GP. Thank you for listening. I hope you enjoyed my presentation.