GenChem Week 10 Patient Project: Kinetic tests Today’s Agenda:  Review patient project.  Review kinetic clinical tests.  Develop the day’s problem.  Perform the challenge: determine CK and LD activity in sample.

GenChem The Patient Project  Patient case studies  Patient interview data  Urine analysis (test strips)  CK and LD tests  Glucose tests  Ion-selective electrodes  Diagnosis and presentation as a poster

GenChem Testing for lactate dehydrogenase and creatine kinase The question posed to you is “how much of each enzyme is in your samples?” You will use two different tests to get your answer. What do you know about these tests:  How do they work?  What information do they provide?

GenChem Kinetic Enzyme Pre-lab

GenChem Let′s think! Representingenzyme-mediatedreactions

GenChem What determines the rate of this reaction? Kinetic testing With lots of substrate, the rate is only dependent on the amount of enzyme

GenChem What will the data look like? Kinetic testing With lots of substrate the rate is proportional to the [LDH]

GenChem What do you know about this enzyme? How much do you expect to find in plasma and/or urine? What factors might cause this value to vary? Lactate Dehydrogenase

GenChem What do you know about this enzyme? How much do you expect to find in plasma and/or urine? What factors might cause this value to vary? Creatine Kinase

GenChem Let′s think! Measuring Reaction Rates

GenChem In your groups:  Discuss how you can use Beer’s Law to determine the rate of a reaction. Given values for  and b, A can be measured and C calculated. Let′s think! Using Spectroscopy to measure concentrations

GenChem One U/L activity is defined as the amount of enzyme that will catalyze the formation of one micromole (10 –6 mol) of product in one minute. Let′s think! Using Spectroscopy to measure reaction rates Activity: U/L = (6590) X Rate (absorbance/min) A proportionality constant that takes into account the molar absorptivity and rate constant of the reaction.

GenChem Measuring Absorbance In your groups review your responses to the following pre-lab question and come up with a consensus answer. Describe how you expect to go about setting up Logger Pro to make absorption measurements and how to zero the spectrometer against a standard solution. Let′s think!

GenChem Testing a Sample In your groups review your responses to the following pre-lab question and come up with a consensus answer. Using the Summary of Steps given in chapter 11 of the lab book as a guide prepare a set of directions for performing the reaction. Let′s think!

GenChem Divvying up tasks Your group will need data from both procedures on your patient samples. Each procedure is best performed by a pair of students. Decide in your group who is going to do what.

GenChem Performing the reactions safely. CK & LDH test reagents CK & LDH test reagents - The reagents used in these reactions are not dangerous, (unless you drink them). Patient Samples MUST Patient Samples - These are actual human samples. To protect yourself from inadvertent contact you MUST wear gloves while performing these reactions. Liquid Waste disposal Liquid Waste disposal – All reagents (both used and unused) and all patient samples must be disposed of in the “Biohazard Waste” bottle in the waste hood. Solid Waste disposal Solid Waste disposal – All solids that have come in contact with the samples must be disposed of in the blue bucket labeled “Biohazard Waste” found in the waste hood. This includes all pipet tips and cuvets.

GenChem Using the Pipets For this experiment you will be using a Wheaton Socorex 100 uL fixed volume pipette. Your instructor will now demonstrate its use. Pay close attention. Remember that the used tips are biohazard waste.

GenChem Your Challenge Perform the reactions Measure their absorbances. Available resources:  CK test reagent, obtained from instructor.  LD test reagent, you prepare.  automatic pipet.  various glassware.  Spectrometer. Design and implement an experimental procedure! Let′s explore! You have 90 minutes

GenChemWrap-up Determine the CK and LD activities in your patient samples Activity: U/L = (6590) X Rate Remember: the rate is absorbance/minute

GenChem Progress Report You will now complete and submit the progress report covering this week and last. progress report These are to be individual efforts although consultation is highly encouraged. Identification: name, TA name and group members. Urine Test Strip Results: values and what they mean Precision of pH measurement: cite data, especially measured pH values versus the estimated values from the color chart Precision of glucose measurement. Same as pH Effect of fructose & galactose. What was learned? Effect of Bleach. The questions are in the worksheet. CK and LD activity: calculated answer and what can be concluded about patient condition.