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1. determine and justify the urine production rate after consumption of different types of
drinks.
2. prepare the urine samples for microscopic analysis. 3. identify the composition of urine by performing physical and chemical tests. 4. identify the different types of cellular components, casts, crystals and artifacts found in
urinary sediments.
4. The final lab report is to be submitted by the SCTL group leaders (M01 M22).
Materials and Equipment 1. Isotonic water 2. Coffee (Nescafe) without sugar 3. Sugar (50%) solution 4. Drinking water provided in sealed bottles 5. Beakers (50 ml, 100ml) 6. Urine Collecting Containers 7. Urinary test strip 8. Refractometer 9. Light microscope 10. Microscopic slides clean and dry 11. Cover slips 12. Sterile capillary tubes Experimental (Multipurpose Lab) Part I (Urinalysis): To determine urine production rate after consumption of different types of drinking fluids. Methodology
1. The students are divided into 4 groups with each group containing 5 healthy males. 2. Before the start of the experiment, the selected individual is required to empty his bladder
completely, and the urine collected in container provided. Record the urine volume and time collected as in Table 1.
Each drink 500mL from Bottle A Each drink 500mL from Bottle B Each drink 500mL from Bottle C Each drink 500mL from Bottle D
4. The time each male student starts drinking the solution is recorded immediately. The
students will not be informed of the type of drink they consume.
5. Urine samples from each male student are collect 30, 60 and 120 minutes from the time of
drinking the solution and the volume obtained (individually) is measure. This is recorded as during the experiment data in Table 1. Calculate the urine production rate (ml/hr) for each student during the experiment.
6. Identify the types of drinks consumed by each group in bottles A, B, C and D (drinking
water, 50% sucrose solution, isotonic drink and coffee without sugar, not necessarily in that order) consumed.
7. Justify the type of solution each student drinks in their group based on calculation of the
relative urine production rate.
8. Present all processed data in one table only (hardcopy) per SCTL group to send in on
Thursday, 8 February 2012 at 8 am by the group leader. (Marks will only be given for complete presentation of data).
9. The table should include all facts necessary to get the final urine production rate (ml/hr),
including mean, identification of type of drinks and explanation of reason (in columns). Please use Excel spreadsheet to display and do all calculations. Label your table as Table 1.
1. All students are involved in this experiment. Label the urine specimen container provided
with your name. Bring the urine back to the lab for analysis.
2. Record physical characteristics of urine collected (color, turbidity, sediments and odor) in
Table 2 (provided). 3. Using the urinary test strip (dipstick), determine the urine chemical characteristics (pH, glucose, proteins, ketones, hemoglobin, bilirubin, urobilinogen and leucocytes) for urines collected. Record results in Table 2.
4. For specific gravity measurements use both dipstick and refractomer, and compare
readings obtained. Calculate the presence of solids in urine (gram per liter) from all samples (take the last two digits of the specific gravity reading and multiply with 2.66 the Longs Coefficient). For example, if the specific gravity reading is 1.025, the solid content is calculated as 25 X 2.66 = 66.5 g/L. Record data obtained in Table 2, and compare these results with the two methods used (dipstick and refractomer).
Part 3. (Microscopy examination). To determine abnormalities associated with type of casts and crystals found in the urine The first thing you have to do for the microscopic exam is prepare the sediment well based on the following protocol: 1. Collect urine from 6 urine samples from the male students. Place 10ml of well-mixed freshly voided urine in a conical centrifuge tube provided. Start centrifugation between 2,000 and 3,000 rpm for 5 10 minutes, or until a pellet formed at the bottom of the tube. 2. Discard the supernatant until about 0.5 to 1ml is left, and re-suspend the sediment by flicking the bottom of tube gently with the forefinger. Using a clean capillary tube, transfer a small amount to a freshly cleaned and dry microscope slide. DO NOT share capillary tubes and always use a fresh one for every slide. Discard used capillary tubes in the container provided. Put a cover slip on the slide.
3. Observe the sediment under microscope at low (to identify casts), then high power objectives
(X10 X40 X100). For better results, close the condenser way down and reduce the light in order to have the best contrast. Identify and do an estimation of the following urinary sediments:
Sediment
Type ammonium biurate, uric acid, bilirubin, sodium urate, calcium carbonate, cholesterol, calcium oxalate, cystine, calcium phosphate, sulfonamide, leucine, tyrosine, triple phosphate, amorphous crystals (phosphate or urate) Hyaline, cellular, granular, waxy casts, fine and coarse granular casts and artifacts Bacteria- Coccus, bacilli, chains, clusters or singles
Estimation
Crystals
Present or absent
Casts
Present or absent
Microorganisms
Present or absent < 5 cells = + 5 10 cells = ++ > 10 cells = +++ < 5 cells = + 5 10 cells = ++ > 10 cells = +++ Present or absent
Epithelial cells
Transitional or squamous
Others
Results should agree within + 10% or + 2 cells, whichever is larger. Observations should be carried out by at least two persons. Methodology: Part III. (Identification) There will be slides show (OSPE style) during the laboratory time. The slides are photographs taken from microscopic examinations of urine sediments.
G p
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Time (minutes)
Group Total volume of urine(ml) Final Urine Production (ml/hr) Type of fluid consume Justification
rate
1 516ml(second highest) (150+235+131)ml / 2hr =258ml/hr Isotonic Isotonic solution contain water and electrolyte, it is much likely like water, does it effects just like water but much lesser than water.
2 432ml(least) (167+125+140)ml / 2hr =216ml/hr Coffee without sugar Coffee is a diuretic agent. This group supposed to get the most urine production, but they get the least urine production maybe because the students were dehydrated, thus drinking coffee is such drinking hypertonic solution, where increase the osmolarity of ECF. Body compensate by increase ECF volume, thus decrease urine output.
3 493.5ml(third highest) (132+163.5+198)ml / 2hr =246.8ml/hr Sugar (50%) solution This is a hypertonic solution. It increased the osmolarity of ECF. Homeostasis occurs by retaining water, thus decrease urine output.
4 656ml(highest) (244+225+187)ml / 2hr =328ml/hr Water Water increased the volume of ECF and decreased osmolarity. To compensate, it increase urine output.
1. Which of the groups produced the most urine at 30, 60 and 120 minutes and which the least?
What could cause the difference, if any? Urine produced/ time (minutes) Most Least 30 4 3 60 1 2 120 3 1
From the table it shows at first 30 minutes, group 4 produced the most urine, while group 3 produced the least urine. On the 60 minutes, group 1 produced the most urine, while group 2 produced the least urine. On 120 minutes, group 3 produced the most urine, while group 1 produced the least urine. There are few factors that cause difference in urine output. Firstly in this experiment, the amount and concentration of fluid consume. Secondly, the amount of ADH and aldosterone circulating in body. Thirdly, the health of students.
2. Is there any difference in the urine volume produced by each experimental group at 30, 60 and
120 minutes? (Effect of time on same treatment group). Justify your result. Yes. At 30 minutes, the highest urine output is produced by Group 4, followed by Group 2, Group 1 and lastly Group 3. At 60 minutes, the highest urine output is produced by Group 1, followed by Group 4, Group 3 and lastly Group 2 At 120 minutes, the highest urine output is produced by Group 3 followed by Group 4, Group 2 and lastly Group 1 After consuming each fluid kidney will try to regulate the volume and osmolarity in the ECF. For example, group 4 which consuming water will have high volume and low osmolarity. For the first 30 minutes, it has increased in GFR thus increase in urine output, while group 3 which consuming glucose (50%) solution will have high osmolarity by low volume. The kidney will regulate the osmolarity and volume in ECF by retaining water in ECF, thus decrease urine output. The mean of urine production is reduced time by time as regulation in the kidney proceed to maintain the volume and osmolarity of ECF.
3. Is there any difference in the urine volume produced by each experimental group at 30, 60 and
120 minutes? (Effect of drinking solution at each time interval). Justify your result.
Yes. At 30 minutes, the highest urine output is produced by Group 4, followed by Group 2, Group 1 and lastly Group 3. At 60 minutes, the highest urine output is produced by Group 1, followed by Group 4, Group 3 and lastly Group 2 At 120 minutes, the highest urine output is produced by Group 3 followed by Group 4, Group 2 and lastly Group 1 There are differences in the urine volume produced effecting by type of solution in each time interval.
A human body regulates its' internal environment within narrow limits. This is called homeostasis. When the body consumed the fluid, the brain will detect the concentration of solution and stimulate the aldosterone to produce more or less anti-diuretic hormone (ADH), depending on the concentration and osmolarity of the blood. ADH is a hormone that maintains the osmolarity of the blood by controlling the permeability of the walls of the collecting duct, thus enabling the regulation of the amount of water reabsorbed from the glomerular filtrate back into the blood capillaries.
4. Compare the rate of urine production in different experimental group. Explain your result. Group Final Urine Production rate (ml/hr) 1 (150+235+131)ml / 2hr =258ml/hr 2 (167+125+140)ml / 2hr =216ml/hr 3 (132+163.5+198)ml / 2hr =246.8ml/hr 4 (244+225+187)ml / 2hr =328ml/hr
From the table above, group that consume coffee (group2) has the lowest rate of urine production followed by group 3 (glucose), group 1 (isotonic) and lastly group 4 (water). The student who consumes the coffee will produce more ADH to lower his/her blood concentration thus, a lower rate of urine formation will be obtained. On the other hand, when a student consumes a low amount of solute such as the water or an isotonic drink, his/her brain will detect the drop in blood-sugar concentration and inhibits the production of ADH by the aldosterone, making the wall of the collecting duct to be more impermeable to water thus, more water will be loss through the urine, increasing the student's rate of urine formation.
5. How is rate of urine produced affected by the type of drinks consumed in this experiment?
Rate of urine produced is affected by concentration of fluid consume as homeostasis will control the rate of urine formation to maintain an optimum volume and osmolarity of ECF. Isotonic 100 plus Slight increase/normal in ECF volume and osmolarity which does not disturb normal reabsorption and secretion normal urine output normal urine production rate Diuretic effect increase impermeability tubular membrane to water decrease in water reabsorption increase urine output increase urine production rate. *In this experiment, the students might be dehydrated, therefore the diuretic effects of the coffee only appear in the first 30 minutes of the experiment. Increase in ECF osmolarity, decrease ECFvolume decrease GFR stimulates release of renin and release of ADH increase water reabsorption decrease urine output decrease urine production rate Increase ECF volume, decrease ECF osmolarity increase GFR stimulates the release of ANP decrease secretion of ADH Decrease water reabsorption increase rate of urine output.
Water
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6. How is the urine production regulated? Relate the mechanism to the volume of urine produced by
subjects in each experimental group.
Urine production is regulated through osmolarity and volume of extracellular fluid via ADH and aldosterone. Group 1 and group 4, which consume isotonic 100 plus and water, contain less glucose, less concentrate. The brain will detect the drop in blood-sugar concentration and inhibits the production of ADH by the aldosterone, making the wall of the collecting duct to be more impermeable to water thus, more water will be loss through the urine, increasing the student's rate of urine formation. While, for group 2 and group 3 which consume coffee and glucose solution, cause high ECF volume but not ECF osmolarity. This will increase the production of ADH to increase the permeability of distal tubule and collecting duct in the presents of aquaporin-2, thus increase water reabsorption thus decrease urine production
7. Explain the mechanism involved in the production of urine at 30, 60 and 120 minutes. The formation of urine depends on 3 processes, which are filtration, reabsorption and secretion. Urinary excretion rate = Filtration rate Reabsorptionrate +Secretion rate Filtration The blood is filtered by nephrons, the functional units of the kidney. Each nephron begins in a renal corpuscle, which is composed of a glomerulus enclosed in a Bowman's capsule. Cells, proteins, and other large molecules are filtered out of the glomerulus by a process of ultrafiltration, leaving an ultrafiltrate that resembles plasma (except that the ultrafiltrate has negligible plasma proteins) to enter Bowman's space. Filtration is driven by Starling forces. The ultrafiltrate is passed through, in turn, the proximal convoluted tubule, the loop of Henle, the distal convoluted tubule, and a series of collecting ducts to form urine. Reabsorption Tubular reabsorption is the process by which solutes and water are removed from the tubular fluid and transported into the blood. It is called reabsorption (and not absorption) because these substances have already been absorbed once (particularly in the intestines). Reabsorption is a two-step process beginning with the active or passive extraction of substances from the tubule fluid into the renal interstitium (the connective tissue that surrounds the nephrons), and then the transport of these substances from the interstitium into the bloodstream. These transport processes are driven by Starling forces, diffusion, and active transport. Indirect reabsorption In some cases, reabsorption is indirect. For example, bicarbonate (HCO3-) does not have a transporter, so its reabsorption involves a series of reactions in the tubule lumen and tubular epithelium. It begins with the active secretion of a hydrogen ion (H+) into the tubule fluid via a Na/H exchanger. In the lumen
Feb 2/CLASS 2012/SEM4/YR2/MHN
The H+ combines with HCO3- to form carbonic acid (H2CO3) Luminal carbonic anhydrase enzymatically converts H2CO3 into H2O and CO2
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Hormones
Cytoplasmic carbonic anhydrase converts the CO2 and H2O (which is abundant in the cell) into H2CO3 H2CO3 readily dissociates into H+ and HCO3HCO3- is facilitated out of the cell's basolateral membrane
Some key regulatory hormones for reabsorption include: aldosterone, which stimulates active sodium reabsorption (and water as a result) antidiuretic hormone, which stimulates passive water reabsorption
Both hormones exert their effects principally on the collecting ducts. Secretion Tubular secretion is the transfer of materials from peritubular capillaries to renal tubular lumen. Tubular secretion is caused mainly by active transport. Usually only a few substances are secreted. These substances are present in great excess, or are natural poisons.
8. Can urine from a dehydrated person show a reading of 1.010 for specific gravity? Explain your
answer. No. reading 1.010 for specific gravity indicates the person is hydrated. In dehydrated person, it will produce concentrated urine, thus increase specific gravity reading.
9. What cause a false increase in the specific gravity reading without actual increase in osmolality? There are appreciable quantities of larger molecules in the urine, such as glucose, radiocontrast media, or the antibiotic carbenicillin. In these settings, the specific gravity can reach 1.030 to 1.050 (falsely suggesting a very concentrated urine), despite a urine osmolality that may be only 300 mosmol/kg.
11. If a marathon runner is asking for your advice, what would you advice the marathon runner to
drink during the race? Justify your answer. Isotonic. During exercise, electrolyte loose through transpiration, by drinking isotonic drinks electrolytes, to restore bodys supply. Also the isotonic drink will maintain the osmolality of blood so that less urine is produced (making rehydration more effective).
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Conclusion: 12. What can you conclude from this experiment? In this experiment, we can conclude that types of drink can affect urine volume production and rate of production. Hifh concentrated type of drink will produce less urine and concentrated urine (high specific gravity reading) while less concentrated fluid will increase urine production with low specific gravity reading.
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TABLE 2. RESULTS OF URINALYSIS (OWN URINE) Substance Reading time using dipstick (after first dip) 30 40 45 nil 60 60 60 60 60 120 Dipstick 1.025 66.5 6.50 0.30 -ve -ve -ve -ve
Glucose Ketones Specific gravity reading *Amount of solid present (grams per Litre g/L) pH Protein Blood Urobilinogen Nitrite Leukocytes
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* multiply the last two digits of reading obtained with Longs Coefficient 2.66 to get amount of solids present in the urine sample.
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Answer the following questions. Q10. Explain the biochemistry (of the urinary strip) for detection of: a. Blood This test is based on the peroxidase-like activity of hemoglobin, which catalyzes the reaction of diisopropylbenzene dihydroperoxide and 3,3',5,5'-tetramethylbenzidine. The resulting color ranges from orange through green; very high levels of blood may cause the color development to continue to blue. The presence of large numbers of RBCs in the urine sediment establishes the diagnosis of hematuria. If the dipstick is more strongly positive than would be expected from the number of RBCs, then the possibility of hemoglobinuria or myoglobinuria should be considered.
b. Glucose
This test is based on a double sequential enzyme reaction. One enzyme, glucose oxidase, 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. In general the presence of glucose indicates that the filtered load of glucose exceeds the maximal tubular reabsorptive capacity for glucose. In diabetes mellitus, urine testing for glucose is often substituted for blood glucose monitoring.
c. Protein
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. Heavy proteinuria usually represents an abnormality in the glomerular filtration barrier. The test is more sensitive for albumin than for globulins or hemoglobin.
d. Ketones
This test is based on the development of colors ranging from buff-pink, for a negative reading, to purple when acetoacetic acid reacts with nitroprusside. Urine testing only detects acetoacetic acid, not the other ketones, acetone or betahydroxybuteric acid. In ketoacidosis (insulin deficiency or starvation), it can be present in large amounts in the urine before any elevation in plasma levels. Q11. What does a positive nitrite test indicate? Positive nitrite indicates bacteriuria containing gram negative bacteria that produce nitrite reductase enzyme which give positive result. Q12. State one possible cause for an individual to produce urine with high pH (> 8.0), excluding the possibility of alkali supplements ingestion. Vegetarian Q13. What further test is needed to confirm the diagnosis stated in Q12? Use pH indicator paper strip
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References 1. Urinalysis http://library.med.utah.edu/WebPath/TUTORIAL/URINE/URIN072.html. 2. Medical Technology, Clinical Laboratory Science Urinalysis http://www.irvingcrowley.com/cls/urin.htm 3. Urinalysis: Pitfalls and Pearls Eileen D. Brewer http://www.baylorcme.org/renal/presentations/brewer/brewer.pdf
4. Continuing Medical Education: Urinalysis Part II. The Urine Microscopic Examination
http://www.medicine.uiowa.edu/cme/clia/modules.asp?testID=20#4_5AbnormalCrystals Submission of laboratory worksheet. Please submit INDIVIDUAL WORK on Table 1 & Table 2, stating objectives of each experiment, and answers to the all questions. This worksheet exercise soft copy must be handed in on Wednesday, 8 February 2012 at 8am to the lecturer (Dr. Mohd Hafiz Ngoo) via Team email. hafizngoo@cybermed.edu.my, and/or mhngoo@yahoo.com
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