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Experiment showing the determination of Creatinine concentration in 24 -hour urine samples. Aim: The aim of the practical is to test the following hypothesis: The amount of creatinine found in a persons 24 -hour urine sample bears a direct relationship to the body mass of that person. METHOD: Materials: 1) 5 urine samples of unknown concentration numbered 1, 5, 6, 7 ad 8. 2) Picric Acid 3) 0. 75 M Sodium Hydroxide 4) P 1000 Gilson Pipette 5) 1 cm 3, 5 cm 3 and 10 cm 3 glass pipettes 6) Vortex Mixer 7) Spectrophotometer 8) Profile 9) Test Tubes Prior to the beginning of the experiment students were give data for the standard curve of creatinine concentration ranging from 0. 05 - 0. 5 mM. Students were also told that creatinine concentrations in a 24 -hour sample are usually between 5 - 20 mM. Therefore the five samples would have to be diluted. PROCEDURE: Dilution factors of x 10, x 20, x 40, x 80 and x 10 diluted sample 1, 5, 6, 7 and 8 respectively.
The x 10 dilution was prepared by taking 1 cm 3 of sample 1 and adding this to 9 cm 3 of distilled water in a clean test tube, this was called the primary procedure. The test tube was labeled 1 d. The x 20 dilution factor was achieved by initially carrying out the primary procedure, however, sample 1 is substituted with sample 5. From this x 10 dilution of sample five 5 cm 3 is removed and added to 5 cm 3 of water in a clean test tube this gave a x 20 dilution the test tube was labeled 5 d. The x 40 dilution was made by initially repeating the primary procedure, however, sample 1 is substituted with sample 6. From this x 10 dilution of sample 6 3 cm 3 is extracted and added to 1 cm 3 distilled water in a clean test tube, this made the x 40 dilution the test tube was labeled 6 d.
First carrying out the primary procedure made the x 80 dilution factor, however, sample 1 was substituted with sample 7. From this x 10 dilution of sample seven 7 cm 3 is removed and added to 1 cm 3 of distilled water. This made the x 80 dilution the test tube was labeled 7 d. The x 100 dilution was prepared by initially executing the primary procedure, however, sample 1 is substituted with sample 8, to give a x 10 dilution of sample 8. 1 cm 3 of this x 10 dilution of sample 8 is removed and added to 9 cm 3 of water in a clean test tube, this gave the x 100 dilution the test tube was labeled 8 d. The above dilutions were carried out using the glass pipettes ensuring a clean set was used for each dilution. The diluted samples were assayed by taking 2 cm 3 of the sample using a p 1000 gilson pipette and placing this in a test tube, then 1 cm 3 of picric acid was added to the test followed by 1 cm 3 0. 75 m sodium hydroxide.
These were then mixed gently but well using the vortex mixer and left to stand for 25 minutes to allow coloured complex to form. All samples were assayed in duplicate therefore the above procedure had to be repeated for each dilution. Along with setting up the above samples a reagent blank had to be prepared this was done in the exact same way as described above however, the 2 cm 3 sample was replaced by 2 cm 3 distilled water. This was also left for 25 minutes. The reagent blank was used to set the reference on the spectrophotometer at 510 nm. Then the absorbance of all the samples was measured at 510 nm.
Once this was completed a calibration graph was drawn using the data for the standard creatinine concentration, 0 05 - 0. 5 mM. The graph was then used to calculate the creatinine concentration in the original urine sample. The values obtained were put into a table. (Attached in results section) RESULTS: To calculate the standard deviation the equation below has to be used. S = The standard deviation % can now be found by dividing the standard deviation value by the mean. After this has been worked out the total amount of creatinine excreted by each individual (in mM) can be calculated, by multiplying the total 24 -hour urine volume (dm 3) by the mean concentration of the eight samples.
SAMPLE Mean creatinine concentration S. Deviation %S. Deviation Concentration of Creatinine (mmol) 1 6. 23 1. 73 27. 8 10. 28 2 14. 26 5. 35 37. 5 10. 84 3 15. 66 4. 24 27. 1 13. 47 4 16. 00 4. 29 26. 8 19. 68 5 11. 52 2. 35 20. 4 9. 91 6 14. 36 4. 89 34. 1 20. 68 7 19. 43 5. 81 29. 9 18. 46 8 9. 55 1. 87 19. 6 13. 66 DISSCUSSION: The results show that there seems to be a relationship between creatinine concentration and body mass. This is supported by graph 1, which shows a linear relationship between creatinine concentration and body mass Graph 1 also shows the correlation coefficient as r = 0. 928, which is very close to 1. 00.
Therefore it is reasonable to say there is a high positive correlation between body mass and creatinine concentration. The second graph displays the standard error bars due to variation; these variations could be due to a number of variables. Firstly the amount of urine supplied could be due to environmental factors, such as the weather if it were a hot the body would retain more water and thus there would be less urine produced, and vice versa for a cold day. The actual weight of the person is also a factor, which can cause variations in the data. The individuals may have been weighed with or without clothes, the scales used to measure the individual may have been inaccurate, or they may have been weighed on soft flooring etc. Another possibility is that the actual sample may not have been a 24 -hour urine sample.
Further errors can be attributed to the student who carried out the experiment. The student may have been inaccurate with their pi petting, or the gilson pipette itself may have been faulty / uncalibrated . The student may have made errors in the calculations regarding the dilutions; this is seen in the large variations in the pooled results. Experimentation al error had to of occurred due to the fact that graph 2 shows a large standard error i. e. the bars are very long, stating a lot of variation.
As variations and errors occurred it is necessary to give remedies or solutions if the experiment were to be carried out again. Firstly the sample set should all be from within the same age group, also when weighing is being carried all members of the sample set should be weighed at the same place using the same scales. To reduce variation that is caused by the weather all members of the sample set should give samples on the same day so the effect of the weather will be the same on everyone. Experimental improvements include greater care when pi petting and more thorough calculations if student is unsure.
Equipment should be high quality and should not under any circumstances be faulty. CONCLUSION: The hypothesis has been proven i. e. the creatinine concentration found in 24 -hour sample of urine is directly related to the mass of a person. SABEEH SYED BSc BIOLOGICAL Sc. 1 st YEAR
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