Laboratory Report No.

5: BODY FLUIDS

Experiments Performed: Part I. Urine Experiment 1: Test for Urine Glucose Experiment 2: Test for Urine Protein Part II. Bile Experiment 3: Test for the Reaction of Bile Experiment 4: Test for the Presence of Bile Pigments Experiment 5: Test for Bile Acids

Date Performed : September 6, 2011

Section A Group 6 Members: 2011-70 2011-70033 2011-70034 2011-70035 2011-70037 2011-70038 BARBOSA, AbIgail CABUANG, Jenel Lyka CAGUITLA, Angela May CAMPO, Ellaine CARLOS, Kim Benedict CASTILLO Suni Grace _________________________ _________________________ _________________________ _________________________ _________________________ _________________________

Introduction

Part I. URINE

Urine is the liquid by-product secreted by the kidneys through a series of filtration, reabsorption, and secretion. Urine formation begins with the process of filtration in the glomerulus. In the glomerulus, blood is under high pressure thus, plasma (except plasma proteins) moves into the glomerular capsule. This fluid, now called the filtrate, contains water, excess salts (Na + and K+), glucose, and urea. Urea is formed in the body to eliminate the very toxic ammonia products that are formed in the liver from amino acids and is the most abundant waste products that must be excreted by the kidneys. The total rate of glomerular filtration rate (GFR) for the whole body (all nephrons in both kidneys) is normally about 125 ml. per minute. That is, about 125 ml. of water and dissolved substances are filtered out of the blood per minute. The next step in urine formation is reabsorption. Reabsorption is the movement of substances out of the renal tubules back to the blood capillaries located around the tubules. This begins at the proximal convoluted tubules and continues to the loop of Henle, distal convoluted tubules, and collecting tubules. Substances that are reabsorbed are water, glucose, other nutrients, sodium, and other ions. Lastly, substances move into the distal and collecting tubules from blood into the capillaries around these tubules through a process called secretion. Secretion moves substances out of the blood and into the tubules where they mix with water and other wastes and are converted into urine to be eliminated. Substances secreted are hydrogen ions, potassium ions, ammonia, and certain drugs. Thus, kidney secretion plays a crucial role in maintaining the bodys acid-base balance. The appearance and smell of your urineas well as the frequency with which you have to go can provide many clues to what else is going on in your body, says Dr. Michael Farber, director of the Executive Health Program at Hackensack University Medical Center in Hackensack, N.J. Several urine tests may give valuable information about ones health and probable diseases. The color, clarity, odor, specific gravity, pH, protein, glucose, nitrites, ketones, and presence of certain particles are often clues that reveal the health status of a person. Glucose is the sugar found in blood and normally, there is very little or no glucose in the urine. In uncontrolled diabetes when the blood sugar is very high, glucose spills over into the urine. Thus, presence of glucose in the urine may indicate diabetes or damaged kidney function. Proteins are also not normally found in the urine. This is because the kidneys are supposed to keep large molecules like protein in the blood. Presence of protein may be due to different factors like fever, hard exercise, pregnancy, and some kidney diseases. A protein urine test measures the amount of protein, such as albumin, in a urine sample. Objectives 1. To test and identify the composition of urine 2. To differentiate normal composition from abnormal composition of urine 3. To test for glucose present in the urine sample 4. To determine the presence of protein in the urine Materials A. Equipment i. Test tubes ii. Alcohol lamp iii. Dropper B. Reagents i. Benedicts reagent ii. 5 % acetic acid C. Urine samples i. ii. iii. iv. Normal Pre-eclampsia Hypertension Diabetes Mellitus (DM)

v. Urinary Track Infection (UTI) Experiment 1: Test for Urine Glucose Methodology: 1 mL of Benedicts reagent was added on a test tube containing 1 mL of urine. The test tube containing the mixture was heated using the alcohol lamp. The color of the solution was observed. Results: Case 1. Normal 2. Pre-eclampsia 3. Hypertension 4. Diabetes Mellitus 5. Urinary Track Infection Discussion: Benedict s test is a general qualitative test used to detect the presence of reducing sugar in a solution. Benedicts reagent contains copper sulfate which when reduced (copper II to copper I) forms a yellow precipitate which is copper oxide. All monosaccharide, including glucose, yield positive result since they possess either aldehyde or ketone group acts as reducing agents. CuSO4
Cu ++ + SO4 --

Glucose (-) (-) (-) (++) (-)

Changes Observed Blue green solution Blue green solution Blue green solution Orange yellow solution Blue green solution

2 Cu++ + Reducing Sugar Cu+

Cu+

Cu2O yellow precipitate

Normal urine does not contain glucose. Almost all glucose that is filtered in the glumorelus is actively reabsorbed in the proximal convoluted tubule. Tubular reabsorption of glucose occurs by active transport in response to the requirement of the body. Hence, for patients with elevated glucose concentration, tubular reabsorption does not occur which is responsible for the presence of sugar in the urine (glycosuria). This explains why in the experiment, the urine specimen obtained from the diabetic patient yielded a positive result in for the Benedict test. The urine specimen from hypertensive patient tested negative for the Benedicts test. Theoretically, it is possible that the condition may cause the presence of glucose in the urine. The increase in blood pressure increases the glomerular filtration rate causing more glucose and other filtrates to to pass through the glomerulus. If the kidneys are intact, despite of the high filtration rate, almost all glucose are still reabsorbed in the proximal convoluted tubule, but for patients with compromised kidney function this may not be true. Pre-eclampsia is a medical condition on which hypertension arises during pregnancy. This condition is usually associated with the presence of protein in the urine, though glucose may also appear in the specimen. The urine specimen of the patient with pre-eclampsia tested negative for the Benedicts test. Lastly, specimen from patient with urinary tract infection (UTI) was also negative since it is usually characterized with the presence of neutrophils or other white bood cells as well as bacteri and not glucose. Conclusion

Benedicts Test detects presence of glucose in urine. A positive test yields a yellow orange solution and this was only observed on the urine specimen obtained from a diabetic patient. Experiment 2: Test for Urine Protein Methodology Urine samples were placed in test tubes (2/3 full). The upper part is heated. The transparency of the heated and unheated portions of the specimen was compared. 5 drops of 5% acetic acid was added on top of the urine without mixing. The same upper portion was heated again. Then the degree of cloudiness present was graded (positive) or labeled negative when the turbidity or cloudiness disappeared. Results Case 1. Normal 2. Pre-eclampsia 3. Hypertension 4. Diabetes Mellitus 5. Urinary Track Infection Discussion 1. Normal urine: (: very slight trace- cloudiness on a dark background) Protein (-) (++) (+++) (-) (++)

The urine of a healthy person normally contains very little protein, usually, less than 10 mg/dL or 100 mg per day is excreted. The protein content consists primarily of low-molecular-weight serum proteins that have been filtered by the glomerulus and proteins produced in the genitorurinary tract. Albumin is the major serum protein found in normal urine. Even though it is present in high concentrations in the plasma, normal urinary albumin is low because majority of the albumin is not filtered into the glomerulus and much filtered albumin is reabsorbed by the tubules. Other proteins present in normal urine include small amounts of serum and tubular microglobulins, Tamm-Horsfall protein produced by the tubules, and proteins from prostatic, seminal and vaginal secretions.

2.

Pre-eclampsia (++: Moderate trace-cloud distinct and granular but no flocculation)

Pre-eclampsia is a hypertension with a renal proteinuria and/ or edema, appearing after 20 weeks of gestation, in labour or within 48 hours of delivery. The result of the urine of a person with preeclampsia was cloudy after heat and acetic acid test. This resulted from increased pressure from blood entering the glomerulus may override the selective filtration of the glomerulus, causing elevated albumin contents in the ultrafiltrate. However, this condition is reversible 3. Hypertension (+++: Heavy cloud marked flocculation with dense cloud)

Urine test can give a lot of information as to how a body is functioning. It can aid in the evaluation of the diseases, especially asymptomatic ones, as indicated by the different substances detected in the urine. For example, having a high blood pressure or hypertension can have fatal effects. Continuous elevation of the blood pressure may eventually damage different organs such as the brain, eyes, heart and kidney. During kidney damage a high protein concentration in the urine is detected. This is because when the kidneys are damaged they are unable to perform their normal function which is to filter blood and retain or reabsorbed filtered proteins and return them to the blood.

4.

Diabetes Mellitus (+++: Heavy cloud- marked flocculation with dense cloud)

A person with either type 1or type 2 diabetes mellitus may lead to reduced glomerular filtration and eventual renal failure is a common occurrence. The urine sample produced marked flocculation with dense cloud. Onset of renal complications can be predicted by detection of microalbuminuria.It could further develop to diabetic nephropathy wherein glomerular membrane is damaged due to its thickening and increased proliferation of mesangial cells and deposition of cellular and noncellular material within the glomerular matrix which results in accumulation of solid substances around capillary tufts. This is associated with deposition of glycosylated proteins, which is caused by poorly controlled blood glucose levels. 5. Urinary Track Infection (++: Moderate trace-cloud distinct and granular but no flocculation)

UTI is an infection of the urinary tract. They filter waste out of the blood and produce urine to carry the wastes and excess water out of the body. The urine is carried from the kidneys, through the ureters, and into the bladder. Urine normally does not have any microorganisms, but if it is obstructed leaving the body or retained in the bladder, it provides an environment for the bacteria to grow. Symptoms differ because it depends on whether the infection affects the lower (bladder and urethra) or upper (kidneys and ureters) of the urinary tract. The kidney infection is more dangerous and can even lead to permanent kidney damage. A urinary tract infection also leads to an infection in the blood stream. Anything that blocks or slows down the way the urine, or introduces bacteria in the urinary tract can raise the risk of having a UTI. The presence of a single type of bacteria at high colony counts is considered positive, but sometimes lower numbers may still indicate infection, especially if symptoms are present. Conclusion Urinalysis is to analyze the content of your urine checks for several substances in your urine, including protein. Kidneys retain the components that the body needs including proteins, while filtering waste products from the blood. However, some conditions allow proteins to pass through the filters of the kidneys, causing protein in urine, but low levels of protein in the urine are normal.

Part II. Bile Introduction Bile primarily consists of organic molecules such as bile acids, cholesterol, phospholipids and many more. They function as an aid for digestion and absorption of fat and fat soluble vitamins in the digestive tract particularly in the small intestine and also functions as means of secretion of many waste products leading to its elimination through the feces. The gall bladder is the organ where bile is modified and stored. An important component of bile is the bile acids that perform a major role during elimination of cholesterol. It is known that cholesterol insoluble in aqueous solutions, but they are made soluble through bile acids. Emulsification of lipid aggregates which makes fat easier and more available for digestion and solubilization and transport of lipids in aqueous environment which is important for transport and absorption of fat soluble vitamins are all made possible by the amphipatic nature of bile acids. Experiment 3: Test for Reaction of Bile Methodology The reaction of bile with litmus paper was tested and changes in the litmus papers color were noted. Results LITMUS PAPER Red Litmus Paper Blue Litmus Paper CHANGES IN COLOR UPON REACTION WITH BILE Turned Blue Remained Blue

Discussion Bile is a yellow, brownish, or olive green digestive fluid made and released by the liver and stored in the gallbladder. It helps down break fats into smaller fatty acids. Bile is partially an excretory product and partially a digestive secretion. It has a pH of 7.6-8.6 which means it is basic. This could explain why the litmus paper turned blue upon reaction with bile. Bile is mainly composed of water, bile salts, cholesterol, a phospholipid called lecithin, bile pigments, and several ions. Experiment 4: Test for the presence of bile pigments Objectives To determine the presence of bile pigments To observe various color changes in Gmelin Test Materials and Methodology Two ml of undiluted bile was allowed to flow down the sides of a test tube containing 5 ml of concentrated HNO3, seeing to it that the two solutions did not mix. The various colors produced at the point of contact were noted. Results At the junction of the two solutions, various colors were observed: green, blue, violet, red, and reddish-yellow. Discussion In the experiment, the Gmelin Test was used to detect the presence of bile pigments. In this test, if bilirubin is present, there is formation of various colors at the junction of the nitric oxide and the sample. The reaction depends on the progressive oxidation of bilirubin to blue-green biliverdin by the addition of nitric acid, changing the yellowish brown pigment to blue and purple; then to green color within seconds (Bruce-Gregorios, 2006). During the stages of oxidation the bilirubin undergoes a series of changes in color which follow the sequence of the familiar solar spectrum (Yeo, 2009). Other bile

pigments present are also oxidized to various colored products aside from biliverdin (green) such as: bilicyanin (blue), bilifuscin (red), and choletelin (yellow) (Rao, 1992). In this test, positive results may not be seen if the concentration of the bile pigments is low in urine (Rao, 1992). Bile pigments detected in the urine suggest jaundice or a liver disease. Conclusion Gmelin Test is used to determine the presence of bile pigments in a solution. Positive results would yield the presence of various colors at the interface of the nitric acid and the sample solution: green, blue, violet, red, and reddish-yellow. Experiment 5: Pettenkofers test for sucrose Objectives: 1. To identify if the bile solution is acidic or basic 2. To determine the presence of different bile pigments using Gmelin and Pettenkofers Tests Materials: test tube 5% diluted bile concentrated H2SO4 sucrose solution

Procedure: 1. In a test tube, 5mL of 5% solution of diluted bile were placed. 2. A 0.5mL of sucrose was added to the tube. 3. The tube was inclined and was slowly poured with 0.5mL of concentrated H 2SO4 without mixing the solution. 4. Color change was observed. Discussion: A color red color was observed after adding sulfuric acid. This is a positive indication of the presence of bile salts. The purpose of sucrose in the experiment was to be converted in fat in order for the bile salt to take action and emulsify it. The sulfuric added after the sucrose will react to the cholic group in the bile solution, thus producing a red colored solution. Conclusion: Bile solution contained bile acids of glycocholic and taurocholic acids. The Pettenkofers test is a way of determining the presence of these acids. A red color change is a positive result after adding sulfuric acid in the bile solution.

References Bruce-Gregorios, J. (2006).Histopathologic Techniques. Quezon City, Philippines: Goodwill Trading Co., Inc. Rao, S. B. (1992).Practical Biochemistry for Medical Students. Calcutta: Academic Publishers. Yeo, G. F. (2009). A Manual of Physiology. London: P. Blakistn, Son & Co. http://www.nlm.nih.gov/medlineplus/ency/article/003580.htm http://www.webmd.com/a-to-z-guides/urine-test http://www.nsbri.org/humanphysspace/focus4/ep-urine.html http://www.ivy-rose.co.uk/HumanBody/Urinary/Urinary_System_Composition_Urine.php Strasinger, S.K. & Di Lorenzo, M. S. (2008).Urinalysis Body Fluids 5th Edition. Philadelphia, PA: F. A. Davis Company. World Health Organization. (2009). Midwifery Education Modules: Education Material for Teachers of Midwifery. World Health Organization. (n.d.). Total bile acids test and clinical diagnosis. http://www.diazyme.com/products/reagents/DZ042A-1.php Retrieved from Diazyme website: