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Urinalysis

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Lab Report
Name: Fathima Nuska
Date: 16/05/2013
Title: Urine Analysis
Introduction:
The urinary system, which is an essential organ system in the body, is most essential for maintaining homeostatic balance in the body. The kidneys being the major organs belonging to the system, involves in removal of metabolic waste products and other foreign materials from the body, thus homeostasis is maintained. The process of filtration and regulation of ions and water, which are the basics of urine formation, starts in the nephrons: the functional units of the kidney.
Explaining in brief, blood flows from the afferent arteriole to the glomerulus. Here plasma is filtrated through a process called glomerular filtration. The filtrate formed, flows to the proximal convoluted tubule from the glomerulus, then proceeds to flow onto the loop of Henley, and then to the distal convoluted tubule. Finally, the filtrate flows into the collecting duct. It has to be taken into note that with the flow of filtrate through the tubules, the filtrate composition changes due to tubular secretion and reabsorption, finally results in the fluid called, ‘urine’.
Urine is defined as a sterile, fluid by-product of metabolic reactions in the body. Since urination is the primary method for excreting toxins, chemicals and drugs from the body, studying a urine sample can provide information about the state and functioning of the body. Analysing the composition and other factors of urine is referred to as Urinalysis and it is a major diagnostic tool used in medicine. A urinalysis can reveal important facts like the state of health and functioning of parts of the body’s excretory system like detecting systemic diseases affecting the kidneys and renal calculi and presence of metabolic disorders in the body and can also detect infections that could be prevailing within the body.
A urinalysis is composed of a gross examination and a chemical examination. Under the gross examination, factors like color, appearance and odor are taken into account while under chemical examination the composition of urine is considered.
Objectives:
To learn the composition and characteristics of urine
To learn the basis of physical and chemical analysis of urine
To learn to perform common physical and chemical examinations on urine
Materials:
Since sets of different experiments were carried out using separate sets of material, the lists of required materials for each of the experiments are given separately under the titles of each of the experiments. 1. Test for proteins * Urine sample * Clean test tube * Dropper * Acetic acid (10%) * Bunsen burner 2. Test for glucose * Urine sample * Clean test tube * Dropper * Benedict’s reagent * Bunsen burner 3. Rothera’s test for ketone bodies * Urine sample * Ammonium sulphate * Sodium nitroprusside * Liquid ammonia * Dropper * Clean test tube 4. Test for bile salts * Urine sample * Clean test tube * Sulfur powder * Spatula

5. Test for Urobilinogen * Fresh urine sample * Ehrlich’s reagent * HCl (20%) * Clean test tube

6. Test for bilirubin * Urine sample * Clean test tube * Barium chloride * Whatman No.1 filter paper * Fouchet’s reagent
Methodology:
Each of the urine samples were labeled as C (referring to the sample used as the control), 1, 2, 3, 4 and 5.
Initially the gross examination was done by observing the color, appearance and the odor of the six provided urine samples. The observations of each of the samples obtained were recorded.
The chemical examination followed next.
Methodology of each of the chemical examinations has been described separately under the respective sub topics.
The first chemical test performed was the test for pH where the pH of the urine sample was detected using a pH meter. The pH meter was placed on a beaker containing water until the neutral pH of 7 was obtained and indicated by the pH meter. Then the pH meter was placed in the first urine sample labeled C. The indicated pH was recorded. Then the pH meter was placed on the rest of the samples 1, 2, 3, 4 and 5 and the indicated values recorded. Care was taken to place the pH meter in the beaker containing water each time before placing it on the sample of urine.
Test for protein:
A clean test tube was filled with urine to 2/3 rd of the tube. Then the upper portion of the urine was boiled using the Bunsen burner for a few minutes. A few drops of 10 % acetic acid was added onto the test tube and heated further. The observation was recorded.
Test for glucose:
5 ml of Benedict’s reagent was taken to a test tube. 8 drops of the urine sample were added onto the same test tube and heated for about 2 minutes. Then the observation was recorded.

Test for ketones (Rothera’s test):
5 ml of urine was taken into a test tube and 5 ml of Ammonium sulphate was also added onto it. Few crystals of Sodium nitroprusside were added and the test tube and shaken well. About 5 ml of liquid Ammonia was taken to a dropper and added along the wall of the test tube. The observation was recorded after sometime.
Test for bile salts:
About 10 ml of urine was taken onto test tube. A little of dry sulfur powder was sprinkled on to the urine and observed and the observation was recorded.
Test for Urobilinogen using Ehrlich’s test:
10 ml of fresh urine samples each were taken into two clean test tubes separately (samples C and 1). 1 ml of Ehrlich’s reagent was added onto one of the test tubes and 20% HCl to the other test tube. Then the test tube was inverted for several times and left to stand for about 5 minutes. After few minutes the color change was observed and recorded.
Test for Bilirubin:
10 ml of urine sample was taken onto a clean test tube. 5 ml of 10% barium chloride was added to the same sample and mixed well. Once sufficient precipitate formed, it was filtered using a filter paper placed upon a funnel. Then the filter paper was unfolded and placed on another dry filter paper. After the solution properly drained away and only the filtrate remained, a drop of Fouchet’s reagent was added onto the precipitate right at the center of the filter paper. The color change was then observed.
Observations:
The observations that were recorded for each of the tests listed above are given below separately:
Observations of the gross examination: Sample | Color | Odor | Description | C | Golden/pale yellow (straw colored) | Inoffensive ammonia smell | Clear, transparent and no debris found, not foamy | 1 | Light orange | Ammonia smell | Slight debris present, clear | 2 | Pale yellow | Ammonia smell | Debris present, not foamy, slightly milky | 3 | Pale yellow | Strong ammonia smell | Opaque, not clear and debris present | 4 | Pale yellow | Ammonia smell; | Less clear, slightly foamy | 5 | Pale yellow | Slight fruity smell | Not foamy, clear |

Observation of pH test: Sample | pH | C | 5.98 | 1 | 7.63 | 2 | 4.65 | 3 | 7.57 | 4 | 5.48 | 5 | 8.43 |

Observations of the protein test:
Samples labeled C and 1 were subjected to the protein test.
Sample C: No change was observed on heating
Sample 1: Upon heating, dense solid formed at the point of heating.
Observations of the glucose test:
Samples 4 and 5 were subjected to the Benedict’s test.
Sample 4: No change was observed
Sample 5: Color turned into brick red
Observations of the ketone test:
Samples 5 and 6 were subjected to the Rothera’s test for ketones:
Sample 5: No change was observed
Sample 6: Purple ring formation at the junction was clearly visible
Observation of the test for bile salts:
Samples 6 and C were subjected to the test for bile salts
Sample 6: Sulfur powder added onto the test tube, floated on the surface of urine
Sample C: Sulfur powder sank onto the bottom of the test tube
Observation of the test for Urobilinogen:
Samples 1 and C were subjected to the test for Urobilinogen
Sample C: Sample turned pink
Sample 1: Sample turned dark red
Observation of the test for bilirubin:
Upon addition Fouchet’s regent, the precipitate turned green.

Discussion:
Urine analysis is an important diagnostic tool used in medicine and generally a complete urinalysis is composed of three components: the gross examination, the chemical examination and the microscopic examination.
Under the gross examination, physical factors like color, odor, texture and appearance are taken into account. Changes in any one or few of these factors indicate abnormalities in the functioning of the body and sometimes can lead to diagnosing different anomalies in the body.
Considering the color of urine, under normal conditions it’s pale yellow (straw colored) in color. Deviating from normalcy sometimes can give rise to colored urine, which can be interpreted as follows: Color | Possibility | Colorless | Diabetes type 1/2, Diuretic or alcohol | Milky | Genitourinary tract diseases | Orange | Fever, Urobilinogenuria | Green | Jaundice, Phenol poisoning | Blue (dirty blue) | Typhus, Cholera | Dark brown ( brown red) | Acute febrile diseases | Black or brown black | Porphyria, Myoglobinuria | Neon yellow | Liver diseases | Red | Trauma |

Colored urine does not necessarily indicate a disease or disorder. At times colored urine can result due to something that was ingested. For example, red urine can be caused by foods like beet or sometimes due to drugs and vitamins like Chloroquine, iron supplements, levodopa, B vitamins, warfarin…etc.
When odor of urine is taken into account, urine of a normal healthy individual is usually an inoffensive ammonical odor while ketosis leads to a slight fruity odor in urine. In addition, bacterial infection can cause the urine to smell very strongly of ammonia.
Further, presence of debris or tiny particulate matter in the urine also can indicate abnormalities in the function of the urinary system.
Under the chemical examination, factors like pH of urine and presence of chemical substances like proteins, glucose, ketones and bile salts in urine are considered.
The normal range of pH of urine is 4.6-8.0(the mean being 6.1), which means it is usually slightly acidic. Usually, a pH below 7 indicates an acidic urine while a pH above 7 indicates an alkaline urine. The pH of urine may sometimes alter due to factors like the diet, some medications, kidney diseases and metabolic diseases like diabetes mellitus. For example, a UTI caused due to bacteria can result in alkaline urine.
Urine + Acetic acid white gelatinous precipitate

Urine + Acetic acid white gelatinous precipitate

When performing the test for proteins, only the upper portion of the urine sample is heated so that the lower portion can be used as a control and be compared with. At an instance where protein is present in urine sample, upon heating the following reaction takes place:

The gelatinous precipitate is due to the presence of albumin.
Based on the extent of the precipitate formed we can derive some conclusions regarding the concentration of protein in the sample. Appearance | Interpretation | No cloudy precipitate | Negative | Barely visible cloudiness | Retest sample | Granular cloudiness | Weakly positive | Dense opaque cloud | Positive (low concentration- 0.2 to 0.3%) | Almost solid, thick precipitate | Positive ( high concentration- greater than 0.5 %) |

Cu2+ Cu+ Cu2+ Cu+ The test for glucose in urine is performed using a chemical named Benedict’s reagent. Benedict’s regent is actually a mixture of Copper sulphate, Sodium carbonate and Sodium citrate. Basically what happens during this test is that the Cu2+ ion is reduced to Cu+ in the presence of glucose which is a reducing sugar, and thus is the reason for the color change from blue to brick red.
(Red)
(Red)
(Blue)
(Blue)

During the color change from blue to red intermediate colors also form, ranging from greenish blue to light red which can indicate the extent of the glucose in urine. Color | Result | Blue to cloudy green | Negative | Yellowish green | Weakly positive | Greenish yellow | Positive | Yellow | Strongly positive (1-2%) | Orange to brick red | Strongly positive (>2%) |

As for the test for ketones in urine, generally, the three following ketone bodies can be detected: Acetone, Aceto acetic acid and β-hydroxybutyric acid. A positive result for this test is assumed with the formation of the purple ring at the junction of the two layers, and presence of ketones in the urine can usually indicate conditions of diabetes, ketosis or starvation in an individual, but it has to be noted that it is not necessarily so.
The test for bile salts in the urine is done using the principle of surface tension. As per the observation, when bile salts are present, sulphur particles added to the sample, sink to the bottom of the test tube. This is because the surface tension of water is reduced due to the presence of bile pigments and thus floatation is prevented. It also has to be noted that, sometimes presence of foam in urine can also indicate bile salts being available in urine. For verification of this, a test tube containing a urine sample can be vigorously shaken and then observed. If foam forms that can indicate bile salts being present too.

For a productive urinalysis to be performed, the initial steps of specimen collection and preservation must be done perfectly.
The urine sample should be collected in a clean, sterile, dry container and should be examined fresh. Generally the technique called the midstream clean catch, where not the first portion but the middle portion of the urine passed is collected, is used for testing.
Though samples taken randomly is enough for the usual tests, at instances, post prandial sample as in the case of test for diabetes and a morning sample as in the case of nephritis are more convenient.
If the urine sample cannot be examined within two hours of collection, it has to be refrigerated. Preservatives like Toluol and formalin too can be used.

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