I. Procedure To begin the exercise, a sample of urine, about 20 mL was taken from one member.

The urine specimen was then examined and the physical characteristics such as color, clarity, odor, etc. were noted. A chemical analysis was followed. It consisted of two parts: the first was for the detection of the presence of glucose, and the second, for the detection of proteins in urine. For the chemical analysis, about 5 mL of the urine specimen is centrifuged at 5-7 rpm. The supernant is set aside for the protein test while uncentrifuged urine specimen was used for the glucose test. To begin the test for the detection of glucose, 3-5 mL of Benedict's reagent was pipetted into a test tube and heated; 5-8 drops of urine was added to the solution while being heated. A change in color was then observed. The test for the detection of proteins in urine required the use of acetic acid as reagent. To begin this, 3-5 mL of centrifuged urine was pipetted into a test tube and the upper portion of the test tube was heated. While heating, the 8 drops of acetic acid was added drop by drop, then the test tube was observed for any changes in consistency or precipitate formation. Results For the physical examination, we were able to observe that the urine specimen taken from our classmate has a pale yellow color, slight turbidity, and very slight odor. The test with the litmus paper indicated that the urine specimen has a 6.5 pH and 1.01 specific gravity. For the chemical analysis of glucose, we weren't able to observe any change in color from blue to orange or brick-red, therefore we can say that the urine specimen was negative for glucose. For the analysis of protein using acetic acid, there was also no formation of precipitate or cloudiness, therefore we can say that the urine specimen was also negative for proteins.

II. Discussion Physical Examination of the Urine The physical characteristics of urine include observations and measurements of color, clarity, odor, specific gravity and pH. Visual observation of a urine sample can give important clues as to evidence of pathology (Complete Urinalysis, n.d.). Color The color of normal urine is usually light yellow to amber. Generally, the greater the solute volume,the deeper the color. The yellow color of urine is due to the presence of a yellow pigment, urochrome. Deviations from normal color can be caused by certain drugs and various vegetables such as carrots, beets, and rhubarb. For example, some people excrete red-colored urine after eating beets. The color is from the natural pigment of beets and is not a cause for worry. Unusual or abnormal urine colors can also be the result of a disease process. For an instance, red-colored urine can occur when blood is present in the urine and can be an indicator of disease or damage to some part of the urinary system. The depth of urine color is also a crude indicator of urine concentration. Pale yellow or colorless urine

indicates a dilute urine where lots of water is being excreted. Dark yellow urine indicates concentrated urine and the excretion of waste products in a smaller quantity of water, such as is seen with the first morning urine, with dehydration, and during a fever (The Visual Examination, n.d.). Odor Urine odor refers to the scent of the fluid excreted during urination. Urine odor varies. Slightly aromatic is characteristic of freshly voided urine. Urine becomes more ammonia-like upon standing due to bacterial activity. Moreover, when a person is dehydrated, his/her urine will have a smell similar to ammonia. However, if one drinks enough fluids and are otherwise healthy, urine does not usually have a strong smell (Complete Urinalysis, n.d.). Changes in urine odor are usually temporary. Such changes are not always a sign of disease. Certain foods and medicines, including vitamins, may affect your urine's odor. For example, asparagus causes a characteristic urine odor due to its content of asparagine, an amino acid. However, foul-smelling urine may be due to bacteria, such as those responsible for urinary tract infections. Sweet-smelling urine may be a sign of uncontrolled diabetes or a rare disease of metabolism. Liver disease and certain metabolic disorders may cause musty-smelling urine (Dugdale, 2011). Clarity Urine clarity refers to how clear the urine is. Usually, laboratories report the clarity of the urine using one of the following terms: clear, slightly cloudy, cloudy, or turbid. "Normal" urine can be clear or cloudy. Substances that cause cloudiness but that are not considered unhealthy include mucus, sperm and prostatic fluid, cells from the skin, normal urine crystals, and contaminants such as body lotions and powders. Other substances that can make urine cloudy, like red blood cells, white blood cells, or bacteria, indicate a condition that requires attention (The Visual Examination, n.d.). pH The kidneys play an important role in maintaining the acid-base balance of the body. Therefore, any condition that produces acids or bases in the body such as acidosis or alkalosis, or the ingestion of acidic or basic foods, can directly affect urine pH. The normal pH of urine ranges from 4.5 - 8.0. Average is 6.0 which slightly acidic. Diet can be used to modify urine pH. A high-protein diet or consuming cranberries will make the urine more acidic. A vegetarian diet, a low-carbohydrate diet, or the ingestion of citrus fruits will tend to make the urine more alkaline.Some of the substances dissolved in urine will precipitate out to form crystals when the urine is acidic; others will form crystals when the urine is basic. If crystals form while the urine is being produced in the kidneys, a kidney stone or calculus can develop. By modifying urine pH through diet or medications, the formation of these crystals can be reduced or eliminated. In addition, bacterial infections also increase alkalinity (Complete Urinalysis, n.d.). Specific Gravity The specific gravity of urine is a measurement of the density of urine - the relative proportions of dissolved solids in relationship to the total volume of the specimen. It reflects how concentrated or dilute

a sample may be. Water has a specific gravity of 1.000. Urine will always have a value greater than 1.000 depending upon the amount of dissolved substances (salts, minerals, etc.) that may be present. Very dilute urine has a low specific gravity value and very concentrated urine has a high value. Specific gravity measures the ability of the kidneys to concentrate or dilute urine depending on fluctuating conditions. Normal range 1.005 - 1.035, average range 1.010 - 1.025. Low specific gravity is associated with conditions like diabetes insipidus, excessive water intake, diuretic use or chronic renal failure. High specific gravity levels are associated with diabetes mellitus, adrenal abnormalities or excessive water loss due to vomiting, diarrhea or kidney inflammation. A specific gravity that never varies is indicative of severe renal failure (Complete Urinalysis, n.d.).

Chemical Analysis of the Urine The use of chemical methods for the analysis of urine allows for the discernment of its contents through chemical reactions. Also, these method allows us to detect materials in the urine that cannot be detected solely by the senses. In the procedure, the chemical tests to be performed were the tests for the presence of glucose and proteins. In most laboratories, prepared dipsticks are used for these tests. The dipsticks are impregnated with reagents needed to react with the desired components. These are dipped into the urine and a corresponding color change will indicate its presence or absence (American Association for Clinical Chemistry, 2009). However, in the actual procedure, manual qualitative tests were employed to perform the chemical analyses. Glucose Glucose is not normally present in the urine because it is a renal threshold substance. This means that it is not excreted in the urine until it reaches certain elevated levels in the blood. Glucosuria or glycosuria is the term used for presence of glucose in the urine (U.S. National Library of Medicine, 2011). To detect glucose, Benedicts test was employed. Benedicts reagent (CuSO4, Na2CO3, Ca3(C6H5O7)2.4H2O) contains the necessary chemicals to carry out the test. This test rests upon the principle that glucose, a reducing sugar, will reduce the cupric ion to cuprous ion. The cuprous ion will be present in solution as cuprous oxide, which is a red precipitate. The CuSO4 shall be the source of cupric ions. The reaction must be carried out in basic conditions and this is provided by Na2CO3 in Benedicts reagent (Nigam, 2007). The equation for the chemical reaction is as follows: Reducing sugar + 2 Cu2+ + 5 OH- --> reduced sugar + Cu2O + 3H2O (red) The red precipitate often appears orange since it is obscured by the blue color of Benedicts reagent. Nevertheless, this would still indicate a positive result. The test solution must be heated as the reaction is favored with the presence of heat. The presence of glucose in the urine may be indicative of certain diseases. One of these could be diabetes, which is caused by low quality or quantity of insulin. However, further tests should be employed

to confirm this disease. This may also be an indicator for pregnancy since gestational diabetes may occur. A rare condition called renal glycosuria may also be possible. This happens when glucose is excreted in the urine even if glucose is at normal levels in the blood (U.S. National Library of Medicine, 2011). Protein Proteinuria is the presence of excess proteins in the urine. Temporarily high level of protein in the urine after exercising, during pregnancy or during an illness is normal, but generally, there should only be small traces of protein in the urine of a healthy individual (Healthwise, 2010). In the experiment, a test using acetic acid and heat was performed for the detection of the protein albumin in the urine. It is based on the principle of heat coagulation and precipitation of protein by the acetic acid resulting to a cloudy solution or presence of white precipitate. It is highly sensitive and can react with many proteins including albumin, globulins, glycoproteins, and Bence-Jones protein (MediaLab, n.d.). The presence of heat and strong acid causes the protein to denaturize and coagulate. This disrupts the hydrogen bonds of the protein structure leaving only its primary structure. It also changes the solubility of the protein wherein the water gets trapped in a semi-solid gel (Raven et.al, 2007). The same principle applies when cooking an egg white (albumin) as seen in the figure below:

Figure 1. Denaturation and coagulation of protein (albumin) When high level of proteins is excreted through the urine, it may be a sign of a kidney disease or abnormality associated with renal failure. However, further quantitative testing of urine for protein may be needed to determine the gravity of proteinuria. When the proteinuria is related to kidney damage, it may be caused by the increase permeability of the glomerular membrane and the loss of negative charges that repel the negatively charge plasma protein in the basement membrane of the glomerular capillaries (Guyton & Hall, 2006). These may be effects of toxic agents, immune complexes found in diseases like lupus erythemaltosus or streptococcal glomerulonephritis. The amount of protein that is present in the urine of an individual with renal failure or damage usually ranges from 10-40 mg/dL (Media Lab, n.d.). Microscopic Examination of Urine Sediment The microscopic elements of the urine sediment is examined to detect and determine possible renal and urinary tract disorders, including other systemic diseases. This test was traditionally performed, but nowadays, it is done only if the preliminary evaluations dictates the need for this test. Criteria from such laboratories determine this.

The microscopic examination of urine sediment is done through centrifugation. The sediment contains red blood cells, white blood cells, epithelial cells, casts, crystals and other structures, which may have accumulated in the urine in its passage from the kidney to the lower urinary tract. High power field or HPF is used to measure the amount of blood cells. 1. Red Blood Cells In fresh urine, red blood cells or erythrocytes (RBCs) have a normal (pale yellow) appearance, appearing as smooth biconcave anucleated disks with 7 micrometers (diameter) and 2 micrometers (thickness). Normal RBCs in urine have <3/hpf. In a hypotonic urine, RBCs swell and lyse, while in a hypertonic urine, they crenate. Dysmorphic RBCs indicate glomerular diseases. 2. White Blood Cells White blood cells or leukocytes (WBCs) can be a sign of infection of other diseases in the kidney. They can enter anywhere to the urine from the glomerulus to the urethra. They are mostly neutrophils approximately 10-12 micrometers (diameter). Normal WBCs in urine have <2/hpf in men and <5/hpf in women. 3. Epithelial Cells

Few renal epithelial cells can be seen in urine microscopic examination due to normal exfoliation. The cells in the upper layer of the epithelium are shed off to give way for new cells. The number of cells should not exceed 15 cells per 10 hpfs (x430) as it is a sign of an active renal disease or tubular injury. There are 3 types of epithelial cells namely renal, transitional and squamous. Renal cells are the cells lining the nephron, glumerulus, proximal and convoluted tubules and the collecting ducts. They are indicative of renal diseases such as acute tubular necrosis, viral infections or renal transplant rejection. Their morphology depends on the origin of the cell, renal cells from the collecting tubules tend to be polyhedral or cuboidal while renal cells derived from the proximal tubules are relatively large, ovoid, or elongated granular cells. Transitional epithelial or urothelial cells are those which line the tract from the renal pelvis to the distal part of the male urethra and to the base of the bladder in females. They are larger and more spherical than renal cells. There is a limited number of transitional cells in a normal person but increases during infection or after urethral catheterization and clumping of these are caused by transitional cell carcinoma. Squamous epithelial cells are those that line the distal portion of male uretha and the urethra, vagina and skin external to the vagina in females. Large amounts of squamous cells in the sample may indicate vaginal contamination in females and foreskin contamination in males. Clue cells, a kind of squamous epithelial cell of vaginal origin, are covered with Gardnerella vaginalis which causes bacterial vaginitis (Ringsrud, 2001). 4. Casts Casts are solidified proteins formed in the lumen of kidney tubules. These are formed in the distal convoluted tubules and the collecting duct of the kidney. Presence of casts in microscopic examinations

indicates existence of a renal disease. There are various different casts all of which has a matrix of TammHorsfall mucoprotein to which other elements may be added. Hyaline casts are solidified Tamm-Horsfall mucoprotein which can be present in healthy individuals. They may increase in numbers after a strenuous exercise and in renal diseases. RBC casts results from the leakage of RBCs in the glomerular membrane or bleeding into the tubules. Presence of RBC casts is indicative of diseases such as vasculitis, acute poststreptococcal glomerulonephtitis and other acute glomerulonephritides, IgA nephropathy or lupus nephritis. It may also result from subacute bacterial endocarditis, renal infarction, and rarely in severe pyelonephritis. WBC casts are composed mainly of neutrophils and indicates an infection in the kidneys. They are often associated with tubulointerstitial disease such as acute pyelonephritis. They may also be seen in acute interstitial nephritis, lupus nephritis, acute papillary necrosis and acute glomerulonephritis. Bacterial casts are bacteria in a protein matrix with WBCs normally present. Bacterial casts are diagnostic of acute pyelonephritis or intrinsic renal infection. Epithelial casts are composed of renal epithelial cells in a protein matrix indicative of a serious pathologic finding. These are associated with acute tubular necrosis, viral disease such as infection with cytomegalovirus, and exposure to nephrotoxic substances such as mercury, ethylene glycol, and various drugs. Waxy casts represent the final stage of cellular degeneration of cells within a cast. They are broad and are probably formed in larger or dilated collecting tubules when there is a significant stasis and tubular atrophy which gave them the name renal failure casts. They are homogenous like hyaline casts but are more refractile, have sharper outlines, blunt ends and fissures along the sides. They associated with severe chronic renal disease and renal amyloidosis, waxy casts are only rarely seen in acute renal disease. Fatty casts are composed of fat globules associated with the nephrotic syndrome. They may also be found in patients with diabetic nephropathy or toxic renal poisoning (Ringsrud, 2001). 5. Crystals Crystals formed in the urine are of limited clinical value. Formation of these does not readily indicates presence of disease but can be due to factors like pH, dehydration, infection and in worst cases, kidney stones (Dion, 1997). Amorphous urates, which are Na, K, Mg or Ca salts, tend to form in acidic urine and have a yellow or yellow-brown color. Amorphous phosphates form in alkaline urine and lack color (Urinary Crystals, n.d.). Elimination via urine of uric acid is approximately 66-75% the quantity depending on the diet. Uric acid crystals are formed in acidic pH less than 5.5. Formation of uric acid crystal is due to excess uric acid or poor dilution in urine (Dion, 1997). Calcium carbonate crystals are large spheroids with radial striations, colorless to yellow-brown. These crystals form in an alkaline pH (Urinary Crystals, n.d.). The aforementioned crystals have little clinical significance and are normally present in urine but unlike these crystals, cystine crystals are found almost exclusively in patients suffering from a genetic disease called cystinuria. Cystinuria impairs the tubular reabsorption of the amino acids lysine, arginine, ornithine and cystine (Dion, 1997).

III. Guide Questions 1. What substances are responsible for the color of the urine? There are several factors that affect the color of the urine. Normally, urine color ranges from pale yellow to dark yellow. This is mainly caused by the substance urobilin, otherwise known as urochrome. Urobilin, the oxidized form of urobilinogen in the urine (Guyton & Hall, 2006), is a yellow biochemical linear tetrapyrrole compound. Similar to bilirubin, urobilin is a degradation byproduct of old blood cells (Urochrome, n.d.). Other factors that can affect the color of the urine are foods, medicines and diseases. Foods like blackberries, beets, rhubarb or blood can cause the red-brown coloring of the blood. Diseases associated to kidneys can make the urine colorless while, vitamin supplements like Vitamin B can make the urine bright yellow (Urine test, 2010). 2. What do the pH and specific gravity of urine indicate? The pH and specific gravity of urine are good indicators of diseases and responses of the body to specific treatments. The normal pH, the acidity or basicity of the urine, ranges from 4.6 to 8.0. Similar to the color of urine, the pH of the urine is also affected by the diet and medications. It can be further affected by systemic acid-base disturbances and renal tubular function. It is commonly manipulated to prevent different types of urinary stones. Some of the pathologic conditions associated to highly basic pH are severe vomiting, kidney diseases, urinary tract infections and asthma. On the other hand, a highly acidic pH is associated to severe lung diseases, uncontrolled diabetes, aspirin overdose and starvation (Urine test, 2010). The specific gravity shows the concentration of solutes in the urine. Normally, a urine sample should have a specific gravity within 1.005 to 1.030. A low specific gravity indicates a low amount of solutes in the urine and high amount of fluids, while high specific gravity indicates the opposite. Severe kidney disease or use of diuretics can be linked to very low specific gravity, while excessive loss of fluids in the form of excessive vomiting, sweating or diarrhea can lead to a high specific gravity of the urine (Urine test, 2010). 3. Give examples of other substances/materials that may be encountered under urine microscopy. During a microscopic urinalysis, substances like red blood cells, white blood cells, casts, crystals, bacteria, yeast cells or parasites, and epithelial cells can be seen. Presence of these substances commonly indicates pathologic conditions. Normally, very few to none blood cells or casts should be seen in the urine. Bacteria, yeast cells, parasites and epithelial cells should neither be present while, few crystals can be seen in the urine (Urine test, 2010). Some pathologic conditions that can be associated to the presence of these substances are hematuria, pyuria, nephrotic syndrome, tubular degeneration, and infections (The University of Utah Eccles Health Sciences Library, n.d.).

4. What happens to the urine specimen if it is not examined within the prescribed period? If the urine specimen is not examined within the prescribed time, it becomes unacceptable for chemical urinalysis as well as microbial testing. There may be potential overgrowth of bacteria which in turn contributes to the disintegration of urine cells and casts. Aside from microbial growth, it is also likely to account for positive error during chemical analysis due to the decomposition of urea to carbon dioxide and ammonia. In turn, this leads to raised pH that induces lysis to the present red blood cells (if ever present) and white blood cells. Other effects are shown below (Mundt & Shanahan, 2011). Table 1. Changes occurring to unpreserved urine Potential Change Color Clarity Odor pH Bilirubin Glucose Ketones Nitrites Urobilinogen Crystals Cells and casts Microorganisms Change Occurring over Time Oxidation of substances Increased turbidity due to proliferation of bacteria Increasing strength due to proliferation of bacteria Increases as bacteria converts urea to ammonia and the loss of CO2 from the specimen Decreasing due to photooxidation and hydrolysis Decreases due to metabolism of organisms Volatilization Increasing due to proliferation of bacteria but also decreasing as the bacteria continue to convert nitrite to nitrogen Decreasing due to oxidation Appearing due to cooling of the specimen Decreasing due to cellular degeneration Increasing due to proliferation

5. Name some health conditions that can produce abnormal findings in the test for glucose and protein. Glucose and protein are two important components of the body. Glucose is the main source of energy while protein is present in glands, bones, organs and others. Both of these components should not be present in the urine which can indicate an abnormal condition in the body. Glucose is normally not present in the urine. Glycosuria is the condition wherein glucose becomes present in the urine. This condition can be caused by a high concentration of glucose in the blood of the patient which is an indication of diabetes mellitus. Another condition signified by glycosuria is the failure of tubules to reabsorb all the glucose in the blood which is called a renal glycosuria ("Patient.co.uk.")Some other conditions that can cause glucosuria include hormonal disorders, liver diseases, medications & pregnancy ("Urinalysis: Three Types of Examinations." ) Protein, unlike glucose, can be present in urine but only in a minimal amount and an elevated concentration also signifies abnormal conditions in the body. Protenuria is a condition wherein the amount of protein in the urine is higher than normal. A kidney disease can be detected with protenuria

and albumin is usually the first type of protein detected. Some disorders that produce high amounts of proteins in the blood are multiple, myeloma, destroyed red blood cells, inflammation, malignancies and an injury in the urinary tract ("Urinalysis: Three Types of Examinations." ).

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Ringsrud, K. (2001, April). Casts in the urine sediment. Laboratory medicine, 32(4), 191-193 Smith, B. and Foster, K. (2013). The urine microscopic: microscopic analysis of urine sediment. Retrieved from http://www.medialabinc.net/urine-microscopic.aspx. The University of Utah Eccles Health Sciences Library. (n.d.) Urinalyis. Retrieved September 18, 2013 from http://library.med.utah.edu/WebPath/TUTORIAL/URINE/URINE.html The Visual Examination (n.d). Retrieved September 18, 2013 from http://labtestsonline.org/understanding/analytes/urinalysis/ui-exams/start/1 Urinary Crystals (n.d.). Animal health diagnostic center. Retrieved September 19, 2013, from https://ahdc.vet.cornell.edu/clinpath/modules/UA-ROUT/crystsed.htm Guyton, A., & Hall, J. (2006). Textbook of medical physiology. (11 ed.). Philadelphia, Pennsylvania: Elsevier Inc. "Urinalysis: Three Types of Examinations." Lab Tests Online: Welcome!. n.p., n.d.Retreive on 18 Sept. 2013 from <http://labtestsonline.org/understanding/analytes/urinalysis/ui-exams/start/1> Urochrome. (n.d.) Retrieved September 18, 2013 from http://www.memidex.com/urochrome Urine test. (2010). Retrieved September 18, 2013 from http://www.webmd.com/a-to-z-guides/urine-test U.S. National Library of Medicine. (2011). Glucose Test Urine. Retrieved September 18, 2013 from http://www.nlm.nih.gov/medlineplus/ency/article/003581.htm.