Experiment 3: Kidney function: Urine Tests Objective: To determine the composition of urine in a normal person and in a patient through

several tests.

Materials and Methods: Refer to Lab Manual

Results: 1. Ammonium Tests Positive control Results Presence of ammonia smell

2. Creatinine (Weyl Test) Creatinine (Weyl Test) Positive control Negative control Sample After NaOH Dark red solution formed Orange solution formed Orange solution formed After Glacial acetic acid Darker yellowish brown solution Lighter yellowish brown solution Lighter yellowish brown solution

3. Uric Acid Test Positive control Negative control Sample Result Dark blue Light green solution Dark Blue

4. Glucose (Benedicts solution) Test Positive control Negative control

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Result Orange solution with precipitate formed Blue solution

Sample

Blue solution

5. MULTISTIX Test Glucose Bilirubin Ketone Specific Gravity Blood pH Protein Urobilinogen Nitrite Leucocytes Indications: -: negative; +: positive Sample 1.010 7.0 Normal Patients urine + 250mg/dL or 14mmol/L 1.005 6.0 >/ 2000 mg/dL or >/20g/L Normal -

Discussion: A urine test can help us to check different components that present in our urine. The test can give information about our health and problems we may have. The kidneys filter the blood and the waste products are passed in the urine. Urine has hundreds of different body wastes and we can perform different tests to determine whether the patient is normal.

Ammonia is generated during the deamination of amino acids in the liver. The normal physiological concentration in blood is less than 35 mol/l. When we were testing the ammonium, the urine does not smell very strong, but has a slightly "nutty" odor. Urine is high in nitrates (compounds high in nitrogen and oxygen). Ammonia is made up of nitrogen and
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hydrogen, NH3. Normally, diluted urine doesn't have much odor. In our urine, ammonia acts as an absorber and leads to water absorption.

Creatinine test measures blood levels of creatinine, a by-product of muscle energy metabolism that filtered from the blood by the kidneys and excreted into the urine. Production of creatinine depends on an individual's muscle mass, which usually fluctuates very little. With normal kidney function, then, the amount of creatinine in the blood remains relatively constant and normal. In this experiment, the positive controls urine was added with a little of a dilute solution of sodium nitroprusside, and then carefully put in a few drops of a weak solution of sodium hydroxide; a dark red color results, changing to darker yellowish brown solution on warming with acetic acid. This may indicate the renal dysfunction. The creatinine clearance rate may be "overestimated" because the active secretion of creatinine will account for a larger fraction of the total creatinine cleared. (Wely, 1851-1913.)

The uric acid urine test measures the amount of uric acid in a sample of urine. Uric acid is produced from the natural breakdown of our body's cells and from the foods we eat. Most of the uric acid is removed from the body in urine while a small portion will be discharged out of the body in form of stool. But if too much uric acid is being produced, the level in the urine will increase. High blood levels of uric acid in the body can cause solid crystals to form within joints. This causes a painful condition called gout. High levels of uric acid in the urine can cause kidney stones. The both results that we had obtained which is sample urine and positive control turn the solution becomes dark blue after the addition of Folin Reagent-Phosphotungstic acid, it is normal since the majority of the uric acid is removed from the body in urine. (Uric Acid in Urine, 20052011)

For us to test the presence of glucose in urine, Benedicts reagent test is performed. The reagent consist mainly copper sulfate and sodium hydroxide. One of the components of Benedict's reagent is copper II oxide when it is placed in solution with glucose (a reducing sugar), the copper II oxide is reduced to copper I oxide ions, and this has a brick red color (OURFOODRECIPES, 2009). The positive control of the test prove that the presence of glucose in urine. A MULTISTIX test can be performed to determine few compounds in urine by placing a special dipstick into a container of a persons urine. The chemicals on the dipstick change color when they come into contact with certain substances.

Sample urine

Patients urine
Figure 1: The figure above shows the result after dipping into different urine.

The sample urine showed all normal results. While the patients urine showed positive results in glucose and protein. Refers to figure 1, the patients urine showed a positive result (as I highlighted in yellow box), it turns the stick into brown yellow in color. When compared to the standard chart (shown below), I estimate the glucose composition that present in patients urine was approximately 500mg/dL or 27mmol/L [Reference range: 140mg/dL]. In normal circumstances, there is very little or no glucose in urine. However, when the blood sugar level is very high in an uncontrolled diabetic patient (exceeds the tubular reabsorptive capacity); sugar might spill over into the urine and glucose can also be found in urine when the kidneys are

damaged or diseased. (Urine Test, 2005-2011). Hence, I have the reason to suspect that this patient is a diabetic patient with glucosuria.

Photo from (Urinalysis, 2011)

This test is based on a double sequential enzyme reaction. One of the enzymes is glucose oxidase, which catalyzes the formation of gluconic acid and hydrogen peroxide from the oxidation of glucose. A second enzyme, peroxidase, catalyzes the reaction of hydrogen peroxide with a potassium iodide chromogen to oxidize the chromogen to colors ranging from green to brown. Generally, the presence of glucose indicates that the filtered load of glucose exceeds the maximal tubular reabsorptive capacity for glucose.

Aside from glucose, the patients urine also shows a positive result on protein by referring to figure 1 (highlighted in red box), the urine turns the stick into aqua in color. When compared to the standard chart (shown below), I estimate the protein concentration that present in patients urine was approximately 300+++ mg/dL or 16.7mmol/L. [Reference range: 150mg/dL]. Protein is normally not found in the urine. Fever, hard exercise, pregnancy, and some diseases, especially kidney disease, may cause protein to be in the urine. (Urine Test, 20052011). The presence of protein may also be a sign for kidney disease. As a result, I have the reason to suspect that the patient may also suffer from proteinuria.

Photo from (Urinalysis, 2011) This test is based on the protein-error-of-indicators (tetrabromphenol blue) principle. At a constant pH, the development of any green color is due to the presence of protein. Colors range from yellow for negative through yellow-green and green to green-blue for positive reactions.
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Heavy proteinuria usually represents an abnormality in the glomerular filtration barrier. The test is more sensitive for albumin than for globulins or hemoglobin.

There were several precautions should be taken while conducting the experiment. Gloves must be worn throughout the entire experiment especially handling with urine samples to prevent any contamination. Besides, in MULTISTIX procedures, the strip should be immersed in the urine sample at least 30 seconds to 1 minute in order for us to examine the color changes accurately. Conclusion: 1. The patient is suspected to be a diabetic patient with excess of glucose and protein in his/her body. 2. The MULTISTIX procedure is much more accurate compared to the conventional method in measuring the components in urine.

References: all-about-diabetes.com. (2007). Retrieved June 18th, 2011, from Normal Sugar Blood Level and Diabetes Charts: http://www.all-about-beating-diabetes.com/normal-sugar-blood-level.html American Academy of Family Physicians. (2004). Retrieved June 18th, 2011, from Impaired Glucose Tolerance and Impaired Fasting Glucose: http://www.aafp.org/afp/2004/0415/p1961.html Ammonia. (2005-2011). Retrieved July 02nd, 2011, from WebMD, LLC.: http://www.webmd.com/digestive-disorders/ammonia-test Chemistry comes alive! . (2001 ). Retrieved June 18th, 2011, from Benedict's Test for Glucose: http://jchemed.chem.wisc.edu/JCESoft/CCA/CCA5/MAIN/1ORGANIC/ORG18/TRAM18/B/05 91308/MOVIE.HTM Creatinine. (2001 - 2011). Retrieved July 01st, 2011, from Lab test online: http://www.labtestsonline.org/understanding/analytes/creatinine/glance.html Creatinine Blood Test. (1996-2011). Retrieved July 2nd, 2011, from MedicineNet, Inc: http://www.medicinenet.com/creatinine_blood_test/article.htm
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Dipstick . (1995-2011). Retrieved July 02nd, 2011, from The Nemours Foundation: http://kidshealth.org/parent/system/medical/dipstick.html Glucose. (2001 - 2011). Retrieved June 18th, 2011, from The Lab Test Online: http://www.labtestsonline.org/understanding/analytes/glucose/test.html Glucose in urine. (1998 - 2011). Retrieved June 18th, 2011, from Sanitoets CC and Sallamander Concepts : http://www.anytestkits.com/utk-glucose-in-urine.htm OURFOODRECIPES. (2009). BENEDICT'S TEST FOR REDUCING SUGARS. Retrieved June 18th, 2011, from BENEDICT'S TEST FOR REDUCING SUGARS: http://benedictstestforreducingsugars.blogspot.com/ Uric Acid in Urine. (2005-2011). Retrieved July 02nd, 2011, from WebMD, LLC: http://www.webmd.com/a-to-z-guides/uric-acid-in-urine Urinalysis. (2011). Retrieved July 02nd, 2011, from Medical Technology : http://irvingcrowley.com/cls/urin.htm Urine Test. (2005-2011). Retrieved July 02nd, 2011, from WebMD, LLC: http://www.webmd.com/a-to-z-guides/urine-test Wely. (1851-1913.). Retrieved July 02nd, 2011, from Weyl, Theodor: http://www.thiemechemistry.com/en/our-service/houben-weyl-centenary/weyl.html