Urinalysis

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Biochemistry Laboratory – CH600 (2008-2009) Experiment 9

Urinalysis *Michelle Dy Sim, Gellina Ann Ram Suderio, Jonnah Kristina Chua Teope  Department of Biology, 3Biology-6, Group # 7, College of Science University of Santo Tomas, España Street, Manila 1008 March 2, 2009 Abstract: Urine is a liquid waste product of the body secreted by the kidneys by kidneys by a process of filtration from blood from  blood called urination and excreted through the urethra. urethra. Urinalysis is an array of tests performed on urine and one of the most common methods of  medical diagnosis. The objectives objectives of this experimen experimentt are to subject subject the urine sample sample acquired acquired to several several tests and to qualitati qualitatively vely examine the presence presence of some normal organic organic constitue constituents nts and pathologic pathologic organic constituen constituents. ts. Initial Initial examination includes the notation of the collection time, color, turbidity, acidity and pH of the urine sample. For the qualitative exam of normal organic constituents, the test for urea, uric acid, indican and creatinine were conducted. In these tests, the patient’s results were all normal. For the qualitative examination for pathologic organic constituents, Gunnings’s test, Benedict’ Benedict’ss test, test, Exton’s test, Smith’s Smith’s test and Occult blood test were performed. performed. The result obtained obtained for the urine sample was that the patient was healthy except for the fact that the patient might have diabetes.

 Keywords: • • •

Urine Urinalysis Laboratory Tests

I. Introduction

Urine is a transparent solution that can range from colorless to amber but is usually pale yellow. Urine is an aqueous solution of metabolic wastes such as urea, urea, dissolved salts, and organic compounds  produced by the kidneys. It plays a vital role in maintaining homeostasis. The production of urine is called diuresis.[1]

Urine ranges from pale yellow to amber because of the pigment urochrome. The color indicates the concentration of the urine and varies with specific gravity. Dilute urine is straw colored, while concentrated urine is deep amber.[2]

The aromatic odor of fresh, normal urine is caused by the presence of volatile acids. Analysis of  the pH of a freshly voided urine specimen indicates the acid – base balance. The urine reflects the work  of the kidneys to maintain normal pH homeostasis.[2] The kidneys maintain maintain normal acid – base balance  primarily through reabsorption of sodium and tubular secretion of hydrogen and ammonium ions.[3]

Urinalysis is part of routine diagnostic and screening evaluati ons performed on urine that provide a general overview of a person’s health.[4] Urinalysis is used as a diagnostic tool because it can help detect detect substa substance ncess or cellul cellular ar materi material al in the urine urine associ associate ated d with with differ different ent metabol metabolic ic and kidney kidney disorders. disorders. It is routinely routinely done in all patients admitted admitted to the hospital, hospital, pregnant pregnant women and presurgical presurgical  patients. It is done diagnostically in patients with abdominal or back pain, dysuria, hematuria, or urinary frequency. It is part of routine monitoring in patients with chronic renal disease and some metabolic diseases.[2]

Most urine tests are performed for one of the following reasons: to diagnose renal or urinary tract disease; to monitor renal or urinary tract disease; to detect metabolic or systematic diseases not directly related to the kidneys.[2]

The process of urinalysis determines the following properties of urine: color, odor, turbidity, specif specific ic gravit gravity, y, pH, glucos glucose, e, ketones ketones,, blood, blood, protei protein, n, bilir bilirubi ubin, n, urobil urobilino inogen gen,, nitri nitrite, te, leukocy leukocyte te estaerase, and other abnormal constituents revealed by microscopic examination of the urine sediment.[3]

This experiment aims to subject the urine sample acquired to several tests and to qualitatively examine the presence of some normal organic constituents and pathologic organic constituents.

II. Methodology A. Initial Examination of Urine

The color, turbidity, acidity and the time when the urine was collected was noted. n oted.

B. Qualitative Examination for Normal Organic Constituents 1.) Test for Urea

A 0.5mL of 70% NaOH and 4 drops of bromine water was added to 1mL urine sample. After, the evolution of N2 gas was observed.

2.) Test for Uric Acid

A 1mL of urine sample was added with 5mL of 20% Na2CO3. Then, 5 drops of phosphotungstic acid reagent was also added then mixed. The formation of a blue color was observed.

3.) Indican Test

To begin with, 5mL of Obemayer’s reagent was added to 5mL urine sample then mixed. Then, 3mL of chloroform was added to it before shaking and allowed to settle. The formation of a blue color in the lower chloroform layer was observed.

4.) Test for Creatinine

A 1mL alkaline alkaline picrate solution solution was added to 2mL urine sample. sample. The formation formation of an orange –  colored solution was noted.

C. Qualitative Examination for Pathogenic Organic Constituents 1.) Gunning’s Test

The 5mL of urine sample was basified with 5 drops of concentrated ammonium hydroxide using a red litmus paper. Then, Lugol’s solution was added to the basic urine, enough to produce a black cloud which does not disappear immediately. It was let stood for 5 minutes. Similar procedure was done for  the positive control. The results obtained were compared.

2.) Benedict’s Test

To start with, 5mL Benedict’s reagent was added to 8 – 10 drops of urine sample in a test tube then mixed. It was then heated in a boiling water bath for 2 – 3 minutes then let stood and allowed to cool. The formation of a precipitate was observed. Similar procedure was done for the positive control. The results obtained were compared.

3.) Exton’s Test

First, First, 3mL of urine sample sample and 3mL of Exton’s reagent were mixed in a test tube. The solution was warmed until cloudiness appeared. Similar procedure was done for the positive control. The results obtained were compared.

4.) Smith’s Test

The 5mL urine sample was placed in a test tube. The test tube was inclined and overlayed with 3mL tincture of alcoholic iodine mixture. Similar procedure was done for the positive control. The results obtained were compared.

5.) Test for Occult Blood

A half spatula guaiac powder was added with 5mL of 95% ethanol in a test tube then mixed. To this mixture, 5mL hydrogen peroxide was added . Then, 5mL of this solution was added to 3mL acidified urine. Similar procedure was done for the po sitive control. The results obtained were compared.

III. Results and Discussion

Urea Urea is the maj major end end prod produc uctt of prot protei ein n nitr nitrog ogen en metabo metabolis lism m in humans humans.. Hepati Hepaticc enzyme enzymess conver convertt ammoni ammoniaa from amino acids to urea.[5] Urea is produced in the liver and excret excreted ed through through the kidney kidneyss in the urine. urine. The circul circulati ating ng levels of urea depend upon protein intake, protein catabolism and kidney kidney functi function. on. Elevat Elevated ed urea urea levels levels can occur occur with with dietary changes, diseases which impair kidney function, liver  diseases, congestive heart failure, diabetes and infections. A  positive result shows the formation of N2 gas or moistening of  the sides of the test tubes used. The urea cycle (also known as the orni ornitthine hine cycl cycle) e) is a cycl cyclee of  biochemical of  biochemical reactions occurring in many animals that produces urea ( NH  NH2)2CO from ammonia (NH3). This This cycl cyclee was was the the firs firstt meta metabo boli licc cycl cyclee discovered. In mammals, the urea cycle takes place only in the liver .[6]

Uric acid is an organic compound of carbon, nitrogen, oxygen and hydrogen with the formula C5H4 N  N4O3. Xanthine Xanthine oxidase oxidase oxidizes oxidizes oxypurines such as xanthine xanthine and hypoxanthine hypoxanthine to uric acid. Uric acid is the final breakdown product of purine catabolism.[2] In most other mammals, the enzyme uricase further oxidizes uric acid to allantoin. Uric acid is a strong reducing agent and a potent antioxidant. In humans humans,, about about half half the oxidant oxidant capacit capacity y of plasma plasma comes from from uric uric acid. acid. Humans Humans produce produce large large quantities of uric acid. In human blood, uric acid concentrations between 3.6mg/dL and 8.3mg/dL are conside considered red normal normal by the Americ American an Medica Medicall Associ Associati ation, on, althoug although h signif significa icantl ntly y lower lower levels levels are common in vegetarians due to a decreased intake of purine – rich meat.[2] Diseases related to elevated uric acid levels are kidney stones, Lesch – Nyhan syndrome, CVDs and diabetes. Uric acid reacts with  phosphotungstic acid to produce allantoin and tungsten blue.

+

H3P W12O40 

+ Tungsten Blue

Indican is an indole produced by bacterial action on an amino acid, tryptophan, in the intestine. Most of the indole is excreted in the feces. The remainder is absorbed, metabolized and excreted as indicant in the urine. In normal urine, the amount of indican excreted is small. It is increased with high  protein diets or inefficient protein digestion. If not digested properly, or if the wrong types of proteins are are inge ingest sted ed,, bowel bowel putre putrefa fact ctio ion n can occur occur.. Prob Proble lems ms with with prot protei ein n dige digest stio ion n can can be caus caused ed by overgrowth of anaerobic bacteria, intestinal obstruction, stomach cancer, low stomach acid, parasitic infections, malabsorptive syndromes, fungal infections, lack of digestive enzymes, or liver problems. Following absorption, indole is converted to 3-hydroxy indole in the liver. Detection of indican in the urine depends upon its decomposition and subsequent oxidation of indoxyl to indigo blue and its absorption into a chloroform layer.

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+ HO 2

2

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+ Indoxyl Sulfuric Acid K 

Creatinine test measures the level of the waste product creatinine in the urine. This test indicates whether the kidneys are working properly. Creatine is formed when food is changed into energy through a process called metabolism. Creatine is broken down into another substance called creatinine by the

addition of strong acid or by alkali or by using enzyme, creatine hydroxylase.[7] If the kidneys are damaged damaged and cannot work normally, normally, the amount of creatinine creatinine in the urine goes down while its level in the blood goes up. Alkaline picrate solution is composed of saturated picric acid and 10% NaOH. Most methods used for creatinine determination are based upon the Jaffe reaction. The Jaffe reaction uses saturated picric acid which oxidizes creatinine in alkali forming creatinine picrate, in which creatinine forms a characteristic orange color when treated with alkaline picrate.[7]

 Gunning’s test checks for ketone bodies in the blood or urine. Ketone bodies are three water –  soluble soluble compounds that are produced produced as by – products products when fatty acids are broken down for energy in the body. The excess presence of ketones in urine is associated with diabetes or altered carbohydrate metabolism.[3] Lugol’s solution consists of 5% iodine and 10% potassium iodide in 85% distilled water  with a total iodine content of 130mg/mL. 1 30mg/mL. A positive result shows a formation of iodoform crystals.

Glucose levels are measured to diagnose diabetes. The normal glucose level in the urine is 180mg/d 180mg/dL. L. Urine Urine glucos glucosee levels levels of 300 – 500mg/d 500mg/dL L are common common with with severe severe untreated untreated diabet diabetes. es. Diabetes results from deficient insulin or decreased sensitivity to insulin. The results of a urine glucose test are abnormal in cases of renal glycosuria and diabetes mellitus.[2]

+ 2Cu

2+

+ 4OH-  Cu2O(solid) + 2H2O

 Normally, protein is not found in urine. This is because the kidney is supposed to keep large molecules in the blood and only filter out smaller impurities. If the kidney is diseased, protein will appear in the urine. Exton’s reagent is 5% sulfosalicylic sulfosalicylic acid in a solution solution of sodium sulfate. A cloudy solution shows the presence of albumin.

Hemoglobin breakdown results in bilirubin production. In the liver, bilirubin is conjugated to an acid to make conjugated bilirubin. Unconjugated bilirubin is water soluble and can be excreted in the

urine. urine. Abnorma Abnormall biliru bilirubin bin values values may indica indicate te anemia anemia,, excessi excessive ve breakdo breakdown wn of RBC, RBC, hepati hepatitis tis,, cirrhosis, obstruction of biliary duct, toxic liver damage and biliary tree obstruction. [3] Tincture of  alcoholic iodine is usually 10% elemental iodine in ethanol. Addtion od a solution of alcoholic iodine to urine produces a green color. The green color indicates the presence of bile. The addition of hydrogen  peroxide to 5mL 95% ethanol and guaiac powder oxidizes the guaiac causing a color change. Heme, a component of hemoglobin found in blood, catalyzes this reaction giving a result in about two seconds. A  blue ring indicates a positive result.[8]

A. Initial Examination of Urine Sample Time collected Early morning Light Color Turbidity Clear  Acidity  Neutral 7 pH B. Qualitative Exam of Normal Organic Constituents Test UREA URIC ACID INDICAN CREATININE

Result With little evolution of white fumes; red litmus paper  Formation of blue – colored solution Formation of blue – colored interface in the lower chloroform layer  Yellow to orange – colored solution

C. Qualitative Exam for Pathologic Constituents Test GUNNING’S TEST (ketone bodies) BENEDICT’S TEST (glucose) EXTON’S TEST (albumin) SMITH’S TEST (bile pigments) OCCULT BLOOD TEST

Positive (+) Control Clou Cloudy dy int interf erface ace wi with crys crysta talls

Olive green cloudy solution with  precipitate Cloudy solution

Urine Sample Ligh Lightt ora orang ngee sol solut utio ion n wi with red  precipitate Clear green solution with some cloudy precipitate No cloudiness

Emerald green – colored solution with no precipitates X

Yellow orange interface; no emerald green color  X

A normal urine specimen should be clear. Cloudy urine may be caused by the appearance of pus, RBCs or bacterias; however, normal urine also may be cloudy because of ingestion of certain foods. Abnormally colored urine may also result from a pathologic condition or the ingestion of certain medicines. Dark yellow urine may indicate the presence of urobilinogen or bilirubin.[2]

The kidneys assist in acid – base balance by reabsorbing sodium and excreting hydrogen. An alkaline pH is observed in a patient with alkalemia. Bacteria, urinary tract infection, or a diet in citrus fruits or vegetables may cause increased urine pH. Alkaline urine is associated with calcium carbonate, calcium phosphate and magnesium phosphate stones. Acidic urine is generally obtained from patients with academia, which can result from metabolic or respiratory acidosis, starvation, dehydration, or a diet high in meat products or cranberries. Acidic urine is associated with xanthine, cystine, uric acid and calcium oxalate stones.[2]

Urine pH becomes alkaline on standing, because of the action of urea – splitting bacteria, which  produce ammonia. The urine pH of an uncovered specimen will become alkaline because carbon dioxide vaporizes from the urine. Dietary factors affect urine pH. Ingestion of large quantities of citrus fruits, dairy products, and vegetables produces alkaline urine, whereas a diet high in meat and certain foods  produces acidic urine.[2]

Protein is a sensitive indicator of kidney function. Normally, protein is not present in the urine  because the spaces in the normal glomerular filtrate membrane are too small to allow its passage. If  glomerular membrane is injured, the spaces in the filtrate become larger, and the protein seeps into the filtrate, then into the urine. If this persists at a significant rate, hypoproteinemia can develop as a result of severe protein loss through the kidneys. This decreases the normal capillary oncotic pressure that holds fluid within the vasculature and causes cau ses severe interstitial edema.[2]

Specific gravity is a measurement of the kidney’s ability to concentrate urine.[3] Specific gravity is used to evaluate the concentrating and excretory power of the kidneys. High specific gravity indicates concentrated urine. Low specific gravity indicates dilute urine. Specific gravity refers to the weight of  the urine compared with that of o f distilled water. Particles in the urine give it weight or specific gravity.[4]

Leukocyte (WBC) esterase is a screening test used to detect leukocytes in the urine. Positive results indicate urinary tract infection. Leukocyte esterase is nearly 90% accurate in detecting WBC in urine.[2]

Like the leukocyte esterase screen, the nitrite test is a screening test for identification of urinary tract infection. Nitrite screening enhances the sensitivity of the leukocyte esterase test to detect urinary tract infection. Nitrite testing is only about 50% accurate in detecting WBCs in the urine.[2]

 Normally, no ketones are present in the urine; however, a patient with poorly controlled diabetes and hyperglycemia may have a massive fatty acid catabolism. The purpose of this catabolism is to   pro provi vide de an ener energy gy sour source ce when when gluc glucos osee cann cannot ot be tran transf sfer erre red d into into the the cell cell beca becaus usee of insu insuli lin n insufficiency. Ketones are the end products of this fatty acid breakdown.[2]

Bili Biliru rubi bin n is a majo majorr cons consti titu tuen entt of bile bile.. If bili biliru rubi bin n excr excret etio ion n is inhi inhibi bite ted, d, conj conjug ugat ated ed hyperbilirubinemia will result. Bilirubin in urine suggests disease affecting bilirubin metabolism after  conjugation or defects in excretion. For screening, elevated urine bilirubin concentration can indicate  previously unsuspected liver injury due to disease, gallstones or drug toxicity. Bilirubin in the urine will color the urine dark yellow or orange.[2]

IV. Conclusion

It comes to the conclusion that the patient is therefore free form any sickness aside from the fact that the patient is already close to having diabetes, and should now be watchful of her sugar level intake.

V. References [1] Urinalysis – Retrieved February 27, 2009 http://en.wikipedia.org/wiki/Urinalysis

[2] Pagana, K. D. and Pagana, T. J. Mosby’s Mosby’s Manual of Diagnostic Diagnostic and Laboratory Laboratory Tests. St. Louis: Mosby, 2002. [3] Fischbach, F. and Dunning, M. B. III. A Manual of Laboratory and Diagnostic Tests. Philadelphia: Williams & Wilkins, 2004. [4] Urea Cycle – Retrieved February 28, 2009 http://en.wikipedia.org/wiki/UreaCycle [5] Harr, R. R. Clinical Laboratory Science Review. Philadelphia: F.A. Davis, 2007. [6] Wallach, Wallach, J. Interpretat Interpretation ion of Diagnostic Diagnostic Tests. Philadelph Philadelphia: ia: Wolters Wolters Kluwer Health/Lippinc Health/Lippincott ott Wiliams & Wilkins, 2007. [7] Urine – Retrieved February 27, 2009 http://en.wikipedia.org/wiki/Urine [8] Lehmann, C. A. Saunder’s Saunder’s Manual of Clinical Clinical Laboratory Laboratory Science. Philadelphia: Philadelphia: W.B. Saunders, Saunders, 1998. [9] Worthley, L. I. G. Handbook Han dbook of Emergency Laboratory Tests . New York: Churchill Livingstone, 1996.